Methods and kits for amplifying and detecting nucleic acids

Abstract

This article provides a method and related kits for denaturing vesicle-mediated target nucleic acid amplification, along with their applications. This method accelerates strand displacement amplification (SEA) reactions by promoting the generation of denaturing vesicles in double-stranded target nucleic acid molecules through rapid temperature-changing thermal cycling. The kits contain specially designed primers and polymerases for performing this method. The methods and kits disclosed herein can be used in various situations, such as the diagnosis of infectious or genetic diseases, sample quality control, and single nucleotide polymorphism (SNP) analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an improved method for denaturing vesicle-mediated target nucleic acid amplification, a dedicated kit, and its applications. Background Technology

[0002] Nucleic acids, which can be divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are fundamental elements of all life forms. DNA carries genetic information and is responsible for encoding amino acids, the basic building blocks of proteins. RNA plays a crucial role in gene encoding, decoding, regulation, and expression. Therefore, nucleic acids have been used as important biomarkers in biological research and medical diagnostics. Nucleic acid amplification technology provides an important theoretical basis for the detection of pathogenic microorganisms, the traceability and authentication of biological materials (such as meat), and other gene detection. Establishing a simple, easy-to-operate, sensitive, and rapid nucleic acid detection method is a major goal in the field of biological detection.

[0003] Since the invention of polymerase chain reaction (PCR), continuously improving the detection efficiency and sensitivity of this technology has been a key focus of research. However, limitations in polymerase kinetics and the heating / cooling rates of thermal cycling instruments have meant that PCR amplification reactions can take one hour or longer to complete. Therefore, improving these two key factors (enzyme system and instrument performance) is crucial to shortening the amplification time. With improvements in enzyme systems and commercially available rapid heating / cooling instruments, PCR amplification time has been reduced from 4 hours in the early days to approximately 1 hour currently. However, there are currently no commercially available instruments that can further shorten the reaction time.

[0004] Currently, commercially available thermal cycling instruments primarily utilize a typical 25-50 μL reaction system for energy transfer, which is the main reason for limiting the energy transfer rate and making it difficult to reduce the inherent reaction time. Therefore, some researchers have begun using infrared lamps, droplet infrared lasers, microwaves, and droplet microwave fields to rapidly transfer and heat energy in liquid samples. However, these non-contact heating methods still have shortcomings in sensitivity and accuracy, thus limiting their application in research. On the other hand, researchers utilize microfluidic technology to reduce the volume of the reaction chamber, thereby increasing the reaction rate by reducing the time spent transferring energy into and out of the sample. For example, new platforms for small-volume PCR—such as droplet PCR—can achieve rapid PCR amplification, but still suffer from low throughput and complex processes. Despite various attempts, methods based on rapid PCR amplification (such as rapid cycling PCR and ultra-rapid PCR) remain limited by the development and commercialization of customized thermal cycling equipment. There are currently no reports on whether optimizing current isothermal nucleic acid amplification techniques can solve these problems.

[0005] In this context, a series of isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP), helicase-dependent isothermal deoxyribonucleic acid (HDA), and denaturing vesicle-mediated strand exchange amplification (SEA), have been developed to replace PCR. LAMP is well-known for its high sensitivity and specificity; however, it is prone to sample contamination, and primer design is difficult, making it unsuitable for detecting highly mutant targets. HDA, on the other hand, requires two enzymes in its reaction system, and the two-enzyme system is prone to non-specific amplification, affecting the interpretation of experimental results. These drawbacks have, to some extent, limited the widespread application of these techniques.

[0006] Denaturation bubble-mediated strand exchange amplification (SEA) is a nucleic acid isothermal amplification technique based on denaturation bubbles mediated by DNA respiration. The reaction requires only one enzyme and a pair of primers. The primers invade the denaturing bubble region generated by DNA respiration, where they extend and replace the original complementary strand under the action of polymerase, thus performing exponential amplification. Patent CN 109136337A describes SEA technology capable of amplifying and detecting strands up to 1.0 × 10⁻⁶. -14 M nucleic acid.

[0007] Based on the existing research, there is still a need to establish high-throughput and stable nucleic acid amplification technologies suitable for traditional laboratories. This invention addresses this technological need. Summary of the Invention

[0008] SEA is a nucleic acid isothermal amplification method. Its principle is that DNA respiration causes the spontaneous formation of denaturation bubbles in double-stranded DNA (dsDNA). Then, a pair of primers invade the denaturation bubble and bind to one of the DNA strands. Under the action of polymerase, the original complementary strand is extended and replaced to produce amplification products. Therefore, this method does not rely on a thermal cycler and can directly use the denaturation bubbles spontaneously formed by the template DNA to initiate the isothermal PCR reaction under constant temperature (simple water bath or metal bath) (Shi et al. "Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification" Chem Commun (Camb) (2016) 4; 52(77):11551-4).

[0009] This invention discloses a significant discovery in improving the SEA (Sequencing Amplification) technology process, which significantly improves the efficiency and rate of the amplification reaction by rapidly varying the reaction temperature, even within a small range of temperature changes. Therefore, this application refers to it as "Rapid SEA". Accordingly, this disclosure provides a novel method for amplifying and detecting target nucleic acids in a sample. In some embodiments, the amplification mixture of the method contains a polymerase, a pair of primers, and a sample; during the reaction, the primers specifically hybridize with the target nucleic acid; then the amplification mixture undergoes multiple thermal cycles between a first temperature and a second temperature, thereby amplifying the sequence of the target nucleic acid molecule via Rapid SEA; and wherein the difference between the first temperature and the second temperature is less than about 30°C. In some embodiments, the amplification mixture of the method contains a polymerase, a pair of primers, and a sample; during the reaction, the primers specifically hybridize with the target nucleic acid; then the amplification mixture undergoes multiple thermal cycles between a first temperature and a second temperature, thereby amplifying the sequence of the target nucleic acid molecule via Rapid SEA; and wherein the difference between the first temperature and the second temperature is less than about 25°C. In some embodiments, the amplification mixture of the method comprises a polymerase, a pair of primers, and a sample; during the reaction, the primers specifically hybridize with the target nucleic acid; the amplification mixture is then subjected to multiple thermal cycles between a first temperature and a second temperature to rapidly amplify the sequence of the target nucleic acid molecule via SEA; and wherein the difference between the first temperature and the second temperature is less than about 20°C. In some embodiments, the method further includes detecting the sequence of the amplification product. In some embodiments, the method further includes diagnostics based on the detection.

[0010] In a particular embodiment, the difference between the first temperature and the second temperature is approximately 10-15°C. In a more specific embodiment, the difference between the first and second temperatures is approximately 10°C, approximately 11°C, approximately 12°C, approximately 13°C, approximately 14°C, or approximately 15°C.

[0011] In some embodiments, the polymerase has an optimal temperature for catalyzing primer extension during PCR. In a particular embodiment, the optimal temperature is within the range of a first temperature ± 6°C. In a particular embodiment, the optimal temperature is within the range of a second temperature ± 6°C. In a particular embodiment, the optimal temperature is between the first and second temperatures.

[0012] In some embodiments, the sequence of the target nucleic acid molecule amplified by this method has a first melting temperature, and wherein the first temperature is within ±5°C of the first melting temperature. In some embodiments, a pair of oligonucleotide primers has an average melting temperature, and wherein a second temperature is within ±5°C of the average melting temperature. In some embodiments, the average melting temperature is within ±5°C of the polymerase's optimal temperature. In some embodiments, one primer in a pair of oligonucleotide primers has a second melting temperature, while the other primer in the pair of oligonucleotide primers has a third melting temperature, wherein the difference between the second and third melting temperatures is less than about 3°C.

[0013] In some embodiments, a first melting temperature is determined using a computer algorithm based on the sequence of the target nucleic acid molecule. Alternatively, in some embodiments, a second melting temperature is determined using a computer algorithm based on the sequence of the oligonucleotide primers. Alternatively, in some embodiments, a third melting temperature is determined using a computer algorithm based on the sequence of the oligonucleotide primers. In some embodiments, the computer algorithm is selected from NUPACK, DNAMelt, NOVOPRO, BLAST, Primer Premier, AlignMiner, Oligo, PerlPrimer, Primer3Web, and DNAstar. In some embodiments, the method further includes determining the first, second, and / or third melting temperatures.

[0014] In some embodiments, the polymerase is a thermostable polymerase. In some embodiments, the polymerase has chain displacement activity. In some embodiments, the polymerase has reverse transcriptase activity.

[0015] In some embodiments, the polymerase is Bst DNA polymerase or an isomer thereof, or a functional derivative having at least 80% sequence identity. In specific embodiments, the polymerase is a large fragment of Bst DNA polymerase or an isomer thereof, or a functional mutant having at least 80% sequence identity. In specific embodiments, the polymerase is a full-length Bst DNA polymerase, a large fragment of Bst DNA polymerase, a Bst 2.0 DNA polymerase, a Bst 2.0 WarmStart DNA polymerase, or a Bst 3.0 DNA polymerase. In specific embodiments, when the polymerase is any polymerase described in this paragraph, a first temperature is in the range of about 68-78°C, and a second temperature is in the range of about 55-69°C.

[0016] In some embodiments, the polymerase is DNA polymerase I or an isomer thereof, or a functional mutant having at least 80% sequence identity. In some embodiments, the polymerase is DNA polymerase I large fragment (Klenow), or an isomer thereof, or a functional mutant having at least 80% sequence identity. In specific embodiments, the polymerase is wild-type DNA polymerase I, DNA polymerase I large fragment (Klenow), or Klenow exo - In a particular embodiment, when the polymerase is any polymerase described in this paragraph, the first temperature is in the range of about 50-60°C, and the second temperature is in the range of about 30-40°C.

[0017] In some embodiments, the polymerase is Vent DNA polymerase or an isoform thereof, or a functional mutant having at least 80% sequence congruence. In specific embodiments, the polymerase is Vent DNA polymerase, Vent(exo-) DNA polymerase, Deep Vent DNA polymerase, or Deep Vent(exo-) DNA polymerase. In specific embodiments, when the polymerase is any polymerase described in this paragraph, a first temperature is in the range of about 70-80°C, and a second temperature is in the range of about 55-70°C.

[0018] In some embodiments, the polymerase is phi29 DNA polymerase or an isomer thereof, or a functional mutant having at least 80% sequence concordance. In particular embodiments, when the polymerase is any polymerase described in this paragraph, the first temperature is selected from the range of about 40-55°C, and the second temperature is selected from the range of about 20-37°C.

[0019] In some embodiments, the polymerase is a Taq DNA polymerase or an isoform thereof, or a functional mutant having at least 80% sequence identity. In specific embodiments, the polymerase is a Taq DNA polymerase, a hot-start Taq DNA polymerase, an EpiMark hot-start Taq DNA polymerase, a OneTaq DNA polymerase, a OneTaq hot-start DNA polymerase, a LongAmp Taq DNA polymerase, or a LongTaq DNA polymerase. In specific embodiments, when the polymerase is any polymerase described in this paragraph, the first temperature is in the range of about 70-88°C, and the second temperature is in the range of about 58-70°C.

[0020] In some embodiments, the length of the amplified sequence and the length of at least one primer are in the range of about 30-60%. In a particular embodiment, the amplified sequence is about 20-50 base pairs (bp) long. In a particular embodiment, the primer is about 15 to about 25 nucleotides (nt) long.

[0021] In some embodiments, the melting temperature (T) of at least one primer m The value is within ±5°C of the polymerase's optimal temperature. In some embodiments, the T of the primer... m The difference between values ​​is less than 1°C. In some embodiments, the G / C content of at least one primer is from about 40% to about 60%. In some embodiments, the difference in the percentage of G / C content between primers is less than 20%. In some embodiments, each primer includes an extension end of a nucleotide that can be added by the polymerase during PCR, and wherein the primer has G or C at the extension end. In some embodiments, each primer includes an extension end of a nucleotide that can be added by the polymerase during PCR, and wherein the primer has at least 40% G / C content in a continuous 5-nucleotide region including the extension end.

[0022] In some embodiments, each thermal cycle includes incubating the amplification mixture at a first temperature for less than 2 seconds and at a second temperature for less than 2 seconds. In some embodiments, each thermal cycle also includes a temperature rise / fall time of less than 10 seconds. In some embodiments, each thermal cycle includes incubating the amplification mixture at a first temperature for about 1 second and at a second temperature for about 1 second, wherein the temperature change time is less than 2 seconds. In some embodiments, the method completes at least 35 thermal cycles in less than 10 minutes, or at least 40 thermal cycles in less than 8 minutes.

[0023] In some embodiments, the amplification mixture further comprises dUTPs. In some embodiments, the amplification mixture does not contain dTTPs. In some embodiments, the amplification mixture further comprises uracil-DNA glycosylase (UDG). In some embodiments, the amplification mixture further comprises single-stranded binding protein (SSB). In some embodiments, the amplification mixture further comprises polyethylene glycol.

[0024] In some embodiments, the amplification mixture contains no more than 1.0 × 10⁻⁶. -12 The target nucleic acid of M. In some embodiments, the amplification mixture contains less than 10 copies of the target nucleic acid. In some embodiments, the amplification mixture contains polymerase at a concentration of not less than 0.1 U / μL. In some embodiments, the amplification mixture contains polymerase at a concentration of not less than 1.0 × 10⁻⁶. -6 At least one primer for M. In some embodiments, the amplification mixture contains at least 0.5% by volume polyethylene glycol. In some embodiments, the amplification mixture contains SSB at a concentration of at least 1 μg / mL. In some embodiments, the volume of the amplification mixture is about 1-30 μL.

[0025] In some embodiments, the thermal cycling step is performed by adding the amplification mixture to a microfluidic device at a heating / cooling rate of at least 10 °C / s. In some embodiments, the target nucleic acid is a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule. In some embodiments, the target nucleic acid is DNA or RNA.

[0026] This disclosure also provides, in another aspect, a method for detecting target nucleic acid molecules in a sample. In some embodiments, the amplification mixture of the method includes a polymerase, a pair of primers, and a nucleic acid sample; during the reaction, the primers specifically hybridize with the target nucleic acid; then the amplification mixture is subjected to multiple thermal cycles between a first temperature and a second temperature to rapidly amplify the sequence of the target nucleic acid molecule via SEA, thereby detecting the amplification product in the amplification mixture; and wherein the difference between the first and second temperatures is less than about 30°C. In some embodiments, the amplification mixture of the method includes a polymerase, a pair of primers, and a nucleic acid sample; during the reaction, the primers specifically hybridize with the target nucleic acid; then the amplification mixture is subjected to multiple thermal cycles between a first temperature and a second temperature to rapidly amplify the sequence of the target nucleic acid molecule via SEA, thereby detecting the amplification product in the amplification mixture; and wherein the difference between the first and second temperatures is less than about 25°C. In some embodiments, the amplification mixture of the method includes a polymerase, a pair of primers, and a nucleic acid sample; during the reaction, the primers specifically hybridize with the target nucleic acid; then the amplification mixture is subjected to multiple thermal cycles between a first temperature and a second temperature to rapidly amplify the sequence of the target nucleic acid molecule via SEA, thereby detecting the amplification product in the amplification mixture; and wherein the difference between the first and second temperatures is less than about 20°C. In specific embodiments, detection is performed every 1, 2, 5, or 10 thermal cycles. In specific embodiments, changes in product yield are detected by detecting the fluorescence signal of the amplification product during the reaction.

[0027] This disclosure also provides, in another aspect, a method for diagnosing pathogen infection in a subject. In some embodiments, the method includes providing a sample containing nucleic acids collected from a subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers specifically hybridize with the nucleic acid of an infectious pathogen; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify a pathogen sequence by polymerase chain reaction (PCR) to detect the presence or absence of an amplification product in the amplification mixture; wherein the difference between the first and second temperatures is less than about 30°C. In some embodiments, the method includes providing a sample containing nucleic acids collected from a subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers specifically hybridize with the nucleic acid of an infectious pathogen; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify a pathogen sequence by polymerase chain reaction (PCR) to detect the presence or absence of an amplification product in the amplification mixture; wherein the difference between the first and second temperatures is less than about 25°C. In some embodiments, the method includes providing a sample containing nucleic acids collected from a subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers specifically hybridize with the nucleic acids of an infectious pathogen; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify the pathogen sequence by polymerase chain reaction (PCR) to detect the presence or absence of amplification products in the amplification mixture; wherein the difference between the first and second temperatures is less than about 20°C. In certain embodiments, the sample contains genomic nucleic acids from the subject or cell-free nucleic acids from the subject. In certain embodiments, the sample is a bodily fluid sample. In certain embodiments, the pathogen is a virus, bacteria, fungus, or parasite.

[0028] This disclosure also provides, in another aspect, a method for detecting genetic alterations in a subject. In some embodiments, the method includes providing a nucleic acid-containing sample collected from the subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers are designed to amplify a target sequence of the subject's genome suspected of containing a genetic alteration; subjecting the amplification mixture to multiple thermal cycles at temperatures between a first temperature and a second temperature to amplify the target sequence by polymerase chain reaction (PCR); wherein the difference between the first and second temperatures is less than about 30°C. The amplified sequence is sequenced to determine the presence of a genetic alteration. In certain embodiments, the genetic alteration is a gene mutation due to nucleotide substitution, deletion, insertion, or copy number variation. In some embodiments, the method includes providing a nucleic acid-containing sample collected from the subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers are designed to amplify a target sequence of the subject's genome suspected of containing a genetic alteration; subjecting the amplification mixture to multiple thermal cycles at temperatures between a first temperature and a second temperature to amplify the target sequence by polymerase chain reaction (PCR); wherein the difference between the first and second temperatures is less than about 25°C. The amplified sequence is sequenced to determine the presence of a genetic alteration. In certain embodiments, the genetic alteration is a gene mutation resulting from nucleotide substitution, deletion, insertion, or copy number variation. In some embodiments, the method includes providing a nucleic acid-containing sample collected from a subject, contacting it with a polymerase and a pair of oligonucleotide primers to form an amplification mixture; wherein the primers are designed to amplify a target sequence of the genome of a subject suspected of containing a genetic alteration; subjecting the amplification mixture to multiple thermal cycles at temperatures between a first temperature and a second temperature to amplify the target sequence by polymerase chain reaction (PCR); wherein the difference between the first and second temperatures is less than about 20°C. The amplified sequence is sequenced to determine the presence of a genetic alteration. In certain embodiments, the genetic alteration is a gene mutation resulting from nucleotide substitution, deletion, insertion, or copy number variation. In certain embodiments, the genetic alteration is a single nucleotide polymorphism. In some embodiments, the method further includes the diagnosis or prognosis of a genetic condition associated with the genetic alteration.

[0029] This disclosure also provides, in another aspect, a kit for performing the method. In some embodiments, a kit for amplifying target nucleic acid molecules is provided. In some embodiments, the kit comprises multiple components, said multiple components including a thermostable polymerase and a pair of oligonucleotide primers, said pair of primers being designed to amplify a target nucleic acid of about 20-50 base pairs (bp) by polymerase chain reaction (PCR). The thermostable polymerase has strand displacement activity.

[0030] In some embodiments of the kit, the melting temperature of at least one primer is within ±5°C of the optimal temperature of the thermostable polymerase. In some embodiments, the G / C content of at least one primer is about 40%-60%. In some embodiments, the difference in G / C content percentage between primers is less than 20%. In some embodiments, each primer includes an extension end to which the polymerase can add nucleotides during PCR, and at least one primer has a G / C content of at least 40% in a continuous 5-nucleotide region at the extension end. In some embodiments, each primer includes an extension end to which the polymerase can add nucleotides during PCR, and at least one primer has G or C at the extension end. In some embodiments, at least one primer is about 15-25 nucleotides in length.

[0031] In some embodiments of the kit, the polymerase is Bst DNA polymerase or an isomer thereof, or a functional mutant having at least 80% sequence identity. In some embodiments, the polymerase is a large fragment of Bst DNA polymerase, or an isomer thereof, or a functional mutant having at least 80% sequence identity. In some embodiments, the polymerase is a full-length Bst DNA polymerase, a large fragment of Bst DNA polymerase, a Bst 2.0 DNA polymerase, a Bst 2.0 WarmStart DNA polymerase, or a Bst 3.0 DNA polymerase.

[0032] In some embodiments, the polymerase is DNA polymerase I or an isomer thereof, or a functional mutant with at least 80% sequence identity. In some embodiments, the polymerase is DNA polymerase I large fragment (Klenow), or an isomer thereof, or a functional mutant with at least 80% sequence identity. In some embodiments, the polymerase is wild-type DNA polymerase I, DNA polymerase I large fragment (Klenow), or Klenow exo - .

[0033] In some embodiments, the polymerase is Vent DNA polymerase or an isoform of it, or a functional mutant having at least 80% sequence congruence. In some embodiments, the polymerase is Vent DNA polymerase, Vent(exo - DNA polymerase, Deep Vent DNA polymerase or Deep Vent (exo) - DNA polymerase. In some embodiments, the polymerase is phi29 DNA polymerase or an isoform of it, or a functional mutant having at least 80% sequence congruence.

[0034] In some embodiments, the polymerase is a Taq DNA polymerase or an isoform thereof, or a functional mutant having at least 80% sequence identity. In some embodiments, the polymerase is a Taq DNA polymerase, a hot-start Taq DNA polymerase, an EpiMark hot-start Taq DNA polymerase, a OneTaq DNA polymerase, a OneTaq hot-start DNA polymerase, a LongAmp Taq DNA polymerase, or a LongTaq DNA polymerase.

[0035] In some embodiments, the kit further comprises dUTPs. In some embodiments, the kit does not comprise dTTPs. In some embodiments, the kit further comprises uracil-DNA glycosylase (UDG). In some embodiments, the kit further comprises a buffer solution suitable for polymerase. In some embodiments, the kit further comprises polyethylene glycol. In some embodiments, the kit also comprises a single-stranded binding protein (SSB), preferably a heat-stable SSB. In some embodiments, the SSB protein is derived from bacteria or bacteriophages. In some embodiments, the SSB protein is selected from T4 bacteriophage 32SSB, T7 bacteriophage 2.5SSB, bacteriophage 29SSB, Escherichia coli SSB, or functional derivatives thereof.

[0036] In some embodiments, the multiple components of the kit are (a) contained in one container, and the kit further includes instructions for adding an appropriate amount of sample to form an amplification mixture; or (b) contained in at least two separate containers, and wherein the kit further includes instructions for mixing the components in the separate containers with an appropriate amount of sample to form an amplification mixture. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.1 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 1.0 × 10⁻⁶. -6 At least one primer for M. In some embodiments, the amplification mixture contains about 0.5%-10% polyethylene glycol by volume. In some embodiments, the amplification mixture contains SSB at a concentration of about 1-50 μg / mL. In some embodiments, the volume of the amplification mixture is about 1-30 μL.

[0037] In some embodiments, the kit further includes instructions for performing PCR using a protocol involving multiple thermal cycles, wherein each thermal cycle includes incubation at a first temperature for no more than 2 seconds, incubation at a second temperature for no more than 2 seconds, and wherein the difference between the first and second temperatures is less than 30°C. In some embodiments, the kit further includes instructions for performing PCR using a protocol involving multiple thermal cycles, wherein each thermal cycle includes incubation at a first temperature for no more than 2 seconds, incubation at a second temperature for no more than 2 seconds, and wherein the difference between the first and second temperatures is less than 25°C. In some embodiments, the kit further includes instructions for performing PCR using a protocol involving multiple thermal cycles, wherein each thermal cycle includes incubation at a first temperature for no more than 2 seconds, incubation at a second temperature for no more than 2 seconds, and wherein the difference between the first and second temperatures is less than 20°C. In specific embodiments, the polymerase is a full-length Bst DNA polymerase, a large fragment of Bst DNA polymerase, Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, or Bst 3.0 DNA polymerase, and wherein the first temperature is in the range of about 68-78°C, and the second temperature is in the range of about 55-69°C. In a particular embodiment, the polymerase is a full-length Bst DNA polymerase, a large fragment of Bst DNA polymerase, a Bst 2.0 DNA polymerase, a Bst 2.0 WarmStart DNA polymerase, or a Bst 3.0 DNA polymerase, wherein each thermal cycle includes incubation for about 1 second at a first temperature selected from a temperature range of about 72-76°C, and incubation for about 1 second at a second temperature selected from about 61-65°C, with a total temperature variation time of less than 2 seconds and a total reaction time of less than 8 minutes.

[0038] In specific embodiments, the polymerase is wild-type DNA polymerase I, DNA polymerase I large fragment (Klenow), or Klenow exo. - The first temperature is in the range of approximately 30-40°C, and the second temperature is in the range of approximately 50-60°C. In a specific embodiment, the polymerase is Vent DNA polymerase, Vent(exo - DNA polymerase, Deep Vent DNA polymerase or Deep Vent (exo) - The method includes a DNA polymerase, wherein a first temperature is in the range of about 70-80°C and a second temperature is in the range of about 55-70°C. In a particular embodiment, the polymerase is phi29 DNA polymerase, wherein the first temperature is selected from the range of about 40-55°C and the second temperature is selected from the range of about 20-37°C.

[0039] In a particular embodiment, the polymerase is Taq DNA polymerase, hot-start Taq DNA polymerase, EpiMark hot-start Taq DNA polymerase, OneTaq DNA polymerase, OneTaq hot-start DNA polymerase, LongAmp Taq DNA polymerase, or LongTaq DNA polymerase, wherein the first temperature is selected from the range of about 70-88°C, and the second temperature is selected from the range of about 58-70°C.

[0040] In some embodiments, each thermal cycle further includes a heating and cooling time of less than 10 seconds. In some embodiments, the number of thermal cycles is less than 40 cycles, and the thermal cycle further includes a total reaction time of less than 10 minutes.

[0041] In some embodiments, the amplification region has a first melting temperature, and the first temperature is within ±5°C of the first melting temperature. In some embodiments, a pair of primers in the kit has an average melting temperature, and a second temperature is within ±5°C of the average melting temperature. In some embodiments, one primer in a pair of oligonucleotide primers has a second melting temperature, while the other primer in the pair of oligonucleotide primers has a third melting temperature, wherein the difference between the second and third melting temperatures is less than about 3°C. Attached Figure Description

[0042] Figure 1 This is a schematic diagram illustrating the principle of denaturation bubble-mediated double-stranded nucleic acid (e.g., DNA) strand displacement amplification reaction.

[0043] Figure 2 This is a real-time amplification curve of the hypervariable target sequence of the Listeria monocytogenes 16S rRNA encoding gene under rapid thermal cycling at 76℃ and 62℃. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different primer concentrations.

[0044] Figure 3 This is a real-time amplification curve of the target sequence of the hypervariable region of the Listeria monocytogenes 16S rRNA encoding gene under rapid thermal cycling at 76℃ and 62℃. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different enzyme concentrations.

[0045] Figure 4This is a real-time amplification curve of the hypervariable target sequence of the 16S rRNA encoding gene of Listeria monocytogenes under rapid thermal cycling at high temperatures between 74℃ and 78℃ and low temperatures between 62℃. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different temperatures.

[0046] Figure 5 This is a real-time amplification curve of artificially synthesized DNA fragments under rapid thermal cycling at 76℃ and 62℃. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction. The Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different target concentrations.

[0047] Figure 6A This is a real-time amplification curve of artificially synthesized RNA fragments under rapid thermal cycling at 76℃ and 62℃. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction. The Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different target concentrations.

[0048] Figure 6B These are polyacrylamide gel electrophoresis (PAGE) images, and the results show... Example 2 The amplification products generated by the rapid SEA reaction described herein. Lane M is a gradient molecular weight DNA ladder, marking the positions of 20 bp and 40 bp DNA bands. The remaining lanes show the specific 43 bp amplification products generated by three replicate control reactions. The initial target concentration for the replicate reactions was 1.0 × 10⁻⁶. -12 M, and no specific amplification products were observed in the negative control. Bands smaller than 20 bp originated from the remaining primer molecules.

[0049] Figure 7A This is a real-time amplification curve of the hypervariable region of the 16S rRNA coding gene of Listeria monocytogenes under rapid thermal cycling at 76℃ and 62℃. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different initial target concentrations.

[0050] Figure 7B These are polyacrylamide gel electrophoresis (PAGE) images, and the results show... Example 3The 43bp amplification product generated by the rapid SEA reaction described herein. Lane M is a gradient molecular weight DNA ladder, and the band positions of 20bp and 40bp DNA are marked. The remaining lanes show the 43bp specific amplification product generated after amplification of the target at different initial concentrations, and no specific amplification product was observed in the negative control.

[0051] Figure 7C This is a real-time amplification curve of the hypervariable target gene of the 16S rRNA encoding gene of Listeria monocytogenes at a constant temperature of 62℃. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction. The Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different initial target concentrations.

[0052] Figure 8 This is a real-time amplification curve of a 50bp fragment encoding the 16S rRNA gene of Staphylococcus aureus under rapid thermal cycling at 76℃ and 61℃. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles performed during the amplification reaction. The Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different target concentrations.

[0053] Figure 9 Descriptions and recommendations from manufacturers for several Bst DNA polymerases are listed, which can be used in conjunction with the methods and kits of the present invention in specific embodiment schemes.

[0054] Figure 10 AE is a real-time amplification curve of the SEA reaction performed at a series of constant reaction temperatures (57℃, 59℃, 61℃, 63℃, and 65℃) using a purified Mycoplasma pneumoniae 16S rRNA encoding gene fragment as a template and five pairs of different primers (Mp1-Mp5). Specifically, Figure 10 A shows the amplification curves of primer pair Mp1 (Tm value approximately 65℃) at five different reaction temperatures. Specifically, Figure 10 B shows the amplification curves of Mp2 (Tm value approximately 63℃) using primer pairs at five different reaction temperatures. Specifically, Figure 10 C represents the amplification curves of Mp3 (Tm value approximately 61℃) using primer pairs at five different reaction temperatures. Specifically, Figure 10 D shows the amplification curves of Mp4 (Tm value approximately 59℃) using primer pairs at five different reaction temperatures. Specifically, Figure 10Figure E shows the amplification curves of primer pair Mp5 (Tm value approximately 57°C) at five different reaction temperatures. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles performed in the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification. The negative control (NTC) results are also shown in the figure above.

[0055] Figure 10 F shows the real-time amplification curves of the SEA reaction performed at five different reaction temperatures, using extracted Mycoplasma pneumoniae genome as a template and Mp3 as the reaction primer. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles performed during the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification. The negative control (NTC) results are also shown in the figure above.

[0056] Figure 11 Figure A shows the real-time amplification curve of the SEA reaction using three specific primer pairs (Ct1-Ct3) with the Chlamydia trachomatis 16S rRNA encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure above.

[0057] Figure 11 Figure B shows the real-time amplification curve of the SEA reaction using three specific primer pairs (Sd1-Sd3) and a domestic pig 18S rRNA-encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure above.

[0058] Figure 12 Figure A shows the real-time amplification curves of the SEA reaction using different specific primer pairs (Mp3, Mp6, and Mp7) and the Mycoplasma pneumoniae 16S rRNA encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles performed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure above.

[0059] Figure 12Figure B shows the real-time amplification curves of the SEA reaction using different specific primer pairs (Ct1, Ct4, and Ct5) with the Chlamydia trachomatis 16S rRNA encoding gene fragment as a template. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles performed in the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification. The negative control (NTC) results are also shown in the figure above.

[0060] Figure 13 Figure A shows the real-time amplification curves of the SEA reaction using different specific primer pairs (Ct1, Ct2, and Ct6) with the Chlamydia trachomatis 16S rRNA encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles performed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure.

[0061] Figure 13 Figure B shows the real-time amplification curves of the SEA reaction using different specific primer pairs (Bc1-Bc3) and the Bacillus cereus 16S rRNA encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles performed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure above.

[0062] Figure 14 This is a real-time amplification curve of the SEA reaction using two specific primer pairs (Sa1 and Sa2) and the Staphylococcus aureus 16S rRNA encoding gene fragment as a template. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. The negative control (NTC) results are also shown in the figure above.

[0063] Figure 15 This is a real-time amplification curve of the SEA reaction accelerated using a microfluidic device. The X-axis represents the amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction. The Y-axis represents the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount. Different symbols represent different target molecule concentrations.

[0064] Figure 16Real-time amplification curves of accelerated SEA reactions using dUTPs or dTTPs are presented. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles performed during the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification. The experiment also includes a negative control group (NTC).

[0065] Figure 17 This is an electrophoretic gel image of the amplification product containing uracil digested by UDG enzyme.

[0066] Figure 18 This is a real-time amplification curve of a rapid SEA reaction using dUTPs with or without UDG enzyme. The X-axis represents amplification time in minutes (min), indicating the number of thermal cycles completed in the amplification reaction, and the Y-axis represents fluorescence signal intensity in relative fluorescence units (RFU), indicating the amplification amount.

[0067] Figure 19 The real-time amplification curves of the target DNA fragment synthesized under rapid thermal cycling between 76°C and 61°C are displayed. The X-axis shows the amplification time in minutes (min), and the Y-axis shows the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification products produced by the reaction.

[0068] Figure 20 The real-time amplification curves of the target sequence in the human β-actin gene are displayed under rapid thermal cycling between 76°C and 61°C. The X-axis shows the amplification time in minutes (min), and the Y-axis shows the fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification products produced by the reaction.

[0069] Figure 21 The figure shows real-time amplification curves of target DNA fragments synthesized under rapid thermal cycling between 76°C and 55°C. The X-axis displays amplification time in minutes (min), and the Y-axis displays fluorescence signal intensity in relative fluorescence units (RFU), indicating the amount of amplification products produced. As shown in the figure, different symbols represent different target concentrations.

[0070] Figure 22 The figure shows real-time amplification curves of target small RNA fragments synthesized under rapid thermal cycling between 60°C and 34°C. The X-axis displays amplification time in minutes (min), and the Y-axis displays fluorescence intensity in relative fluorescence units (RFU), indicating the amount of amplification products produced. As shown in the figure, different symbols represent different target concentrations. Detailed description

[0071] This manual provides methods for amplifying and detecting target nucleic acids in samples.

[0072] This disclosure provides a technical solution for amplifying target nucleic acid. The method involves directly contacting a thermostable polymerase and a pair of oligonucleotide primers with a sample to form an amplification mixture. This amplification mixture is then subjected to alternating thermal cycles at a first temperature of 68-78°C and a second temperature of 55-69°C to amplify the target nucleic acid sequence via polymerase chain reaction (PCR).

[0073] This disclosure, in another aspect, provides a kit for implementing the technical solution. The kit includes, along with a sample, at least one thermostable polymerase and a pair of oligonucleotide primers, as well as instructions for implementing the technical solution using the kit. Given the detailed description of specific embodiments, additional features (as used herein) will be readily understood by those skilled in the art.

[0074] 4.1 General Technology

[0075] The techniques and steps described and involved in this invention are derived from conventional techniques and steps that are easily understood and / or commonly used by those skilled in the art, such as those in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, 2001). Molecular Cloning:A Laboratory Manual (3d ed. 2001) and Ausubel et al., eds., *Experimental Techniques in Modern Molecular Biology* (2003) Current Protocols in Molecular Biology The widely used method described in (Ausubel et al. eds., 2003)).

[0076] 4.2 Terminology

[0077] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. For the purposes of this specification, the following terminology will be used to describe terms, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. In the event of any conflict between any description of terms and any document incorporated herein by reference, the description of terms below shall prevail.

[0078] Unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” used herein include the plural forms.

[0079] As used herein, the term "approximately" means approximately, within a range, roughly, or near. When the term "approximately" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the aforementioned value. Typically, the term "approximately" is used herein to modify a value to be around 5% (±5%) of said value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "approximately," it will be understood that the particular value forms another embodiment. It will also be further understood that each endpoint of a range is significantly related to and independent of the other endpoint.

[0080] The term "amino acid" refers to naturally occurring and non-naturally occurring α-amino acids, as well as α-amino acid analogs and amino acid mimics that function similarly to naturally occurring α-amino acids. Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolidone, and selenocysteine). Amino acid analogs or derivatives are compounds having the same basic chemical structure as natural amino acids, i.e., carbon atoms bound to hydrogen, carboxyl, amino, and side-chain R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methanesulfonic acid. Such analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acids are referred to herein by their well-known three-letter symbols or a one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can be referred to by their commonly accepted single-letter codes.

[0081] As used herein, the term "conservative substitution" refers to the substitution of one amino acid residue by another biologically similar residue. Examples of conservative substitution include the substitution of one hydrophobic amino acid residue, such as Ile, Val, Leu, or Met, for another, or the substitution of one polar amino acid residue, such as between Arg and Lys, between Glu and Asp, or between Gln and Asn, and similar substitutions. In some cases, ionic amino acid residues are substituted by Asp of similar or oppositely charged ionic amino acid residues (e.g., Lys). Such substitutions are considered conserved in the art because those ionic amino acid groups are thought to provide only solubility assistance. The terms "nonionic" and "ionic" amino acid residues are used herein in their usual sense, referring to amino acid residues that are normally uncharged or normally charged at physiological pH values. Exemplary nonionic amino acid residues include Thr and Gln, while exemplary ionic amino acid residues include Arg and Asp.

[0082] The terms "non-natural amino acid," "non-proteinogenic amino acid," or simply "non-natural amino acid" refer to α-amino acids that contain different side chains (different R groups) relative to the twenty-two common or natural amino acids listed above. Furthermore, these terms can also refer to amino acids described as having an L-stereochemical conformation instead of a D-stereochemical conformation, although some amino acids do indeed exist in their naturally occurring D-stereochemical form (e.g., D-alanine and D-serine).

[0083] As used herein, the term "Bst DNA polymerase" refers to a wild-type DNA polymerase derived from *Bacillus stearothermophilus* or a mutant or truncated form thereof that retains at least polymerase and strand displacement activities. This enzyme can be isolated from or synthesized from *Bacillus stearothermophilus*. An exemplary embodiment of the Bst DNA polymerase particularly suitable for this disclosure is Bst DNA polymerase (large fragment), which has been reported to have good strand displacement activity at about 65°C and inherent reverse transcriptase activity (Shi et al. "Innate reverse transcriptase activity of DNA polymerase for isothermal RNA direct detection." J. Am. Chem. Soc. (2015) 137, 13804–13806), and no 5′→3′ exonuclease activity. Other Bst DNA polymerases suitable for the embodiments according to the invention include, but are not limited to, Bst DNA polymerase (full-length), mutant Bst DNA polymerases, such as those from New England Biotech. Commercially available Bst 2.0 DNA polymerase, Bst WarmStart DNA polymerase, and Bst 3.0 DNA polymerase.

[0084] As used herein, the term "functional derivative" of a reference enzyme or protein refers to an enzyme or protein that has a different amino acid sequence than the reference enzyme or protein but retains the same function as the reference enzyme or protein. In some cases, this term is used for one or more activities of interest, and a variant can be considered a functional derivative as long as it retains the activity of interest of the reference, even if the variant may not contain other functions or activities of the reference. In some cases, a functional derivative may maintain the same activity as the reference even if the activity level of the derivative is increased or decreased, and such a derivative can still be considered a functional derivative of the reference.

[0085] In the fields of molecular biology and genetics, the term "G / C content" refers to the percentage of nitrogenous bases guanine (G) or cytosine (C) in a DNA or RNA molecule.

[0086] The term "genetic polymorphism" refers to the phenomenon of two or more DNA sequences coexisting in the same hybrid population.

[0087] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by alignment and comparison of sequences. Sequence identity, defined as the percentage (%) of identical nucleotides in a candidate nucleotide sequence compared to a reference nucleotide sequence, is obtained by aligning sequences and introducing gaps where necessary, to achieve the maximum percentage of sequence identity, without considering any substitutions as part of the sequence identity. Alignments performed to determine the percentage of nucleic acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, exemplary parameters for determining the correlation between two or more sequences using the BLAST algorithm are as follows. In summary, sequence alignment can be performed using BLASTP version 2.0.8 (Jan-05-1999) with the following parameters: matrix: 0BLOSUM62; vacancy open: 11; gap extension: 1; x_dropoff: 50; expectation: 10.0; font size: 3; filter: on. Nucleic acid sequence alignment can be performed using BLASTN version 2.0.6 (September 16, 1998) with the following parameters: match: 1; mismatch: -2; vacancy open: 5; gap extension: 2; x_dropoff: 50; expectation: 10.0; font size: 11; filter: off. Those skilled in the art will understand what modifications can be made to the above parameters to increase or decrease the stringency of the comparison and to determine the relevance of two or more sequences.

[0088] The terms “oligonucleotide” and “nucleic acid” refer to oligomers and polymers of deoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing analogs of known natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to the natural nucleotide. Unless otherwise specifically defined, the term also refers to oligonucleotide analogs including PNA (peptide nucleic acid), DNA analogs (phosphate thioesters, aminophosphate esters, etc.) used in antisense techniques. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by replacing one or more selected (or all) of the three codons with a mixture of bases and / or deoxyinosine residues to produce sequences (Batzer, MA, et al., Nucleic Acid Res., 1991, 19, 5081-1585; Ohtsuka, E. et al., J. Biol. Chem., 1985, 260, 2605-2608; and Rossolini, GM, et al., Mol. Cell. Probes, 1994, 8, 91-98). Nucleic acids as used herein can be, but are not limited to, DNA, RNA, cDNA, gDNA, rRNA, ssDNA, dsDNA, DNA-RNA hybrids, etc.

[0089] As used herein, the term “or” means any single member of a particular list, and also includes any combination of members of that list, unless otherwise stated or implied by the context in which the term appears.

[0090] As used herein, the term "phi29 DNA polymerase" refers to a wild-type replication polymerase derived from Bacillus subtilis phage phi29 (Φ29) or a mutant or truncated form of it that retains at least polymerase and strand displacement activities. This enzyme can be isolated from phage phi29 or synthesized artificially.

[0091] As used in this article, the term “polymerase chain reaction” or PCR refers to a chain reaction catalyzed by a nucleic acid polymerase, in which nucleic acid chains produced in earlier rounds of reaction are used as templates for subsequent reactions.

[0092] As used herein, the terms "probe," "primer," or "oligonucleotide" refer to a single-stranded DNA or RNA molecule with a defined sequence that can pair with the bases of another DNA or RNA molecule containing a complementary sequence (i.e., the "target"). Hybridization is the combination of two complementary single-stranded nucleic acids to form a hydrogen-bonded double strand. The stability of the resulting hybrid depends on its length, G / C ratio, nearest-neighbor stacking energy, and the degree of base pairing. The degree of base pairing is influenced by parameters such as the complementarity between the probe and target molecules and the stringency of the hybridization conditions. The stringency of hybridization is influenced by parameters such as temperature, salt concentration, and concentrations of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides can be detectably labeled using methods well known to those skilled in the art, including radioactive, fluorescent, or non-radioactive labeling. dsDNA-binding dyes (dyes that fluoresce more strongly when bound to double-stranded DNA than when bound to single-stranded DNA or when free in solution) can be used to detect dsDNA. It is understood that "primers" are specifically designed to be extended by polymerases, while "probes" or "oligonucleotides" may not be designed in this way.

[0093] The terms “peptide” and “protein” are used interchangeably herein and refer to polymers of more than fifty (50) amino acid residues. That is, the description of a peptide is equally applicable to the description of a protein, and vice versa. The term applies to native amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-native amino acids, such as amino acid analogs. As used herein, the term includes amino acid chains of more than 50 amino acid residues in length, including full-length proteins (e.g., full-length polymerases) in which amino acid residues are linked by covalent peptide bonds.

[0094] As used herein, the term "peptide" refers to a polymer chain containing two to fifty (2-50) amino acid residues. This term applies to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids, such as amino acid analogs or amino acid polymers containing non-natural amino acids.

[0095] As used herein, the term “sample” means an animal; an animal’s tissue or organ; cells (including cells in a subject; cells taken directly from a subject; cells held in a culture or taken from a cultured cell line); cell lysates (or lysate fractions) or cell extracts; a solution containing one or more molecules (e.g., polypeptides or nucleic acids) derived from cells, cellular material, or viral material; or a solution containing natural or non-natural nucleic acids, as described herein. A sample can also be any bodily fluid or excretion containing cells, cellular components, or nucleic acids (e.g., but not limited to blood, urine, feces, saliva, tears, bile).

[0096] As used herein, the term “specific hybridization” or its grammatical variations refer to a probe recognizing and physically interacting (i.e., base pairing) with substantially complementary nucleic acids (e.g., sample nucleic acids) under highly stringent conditions, and hardly pairing with bases of other nucleic acids. The phrase “highly stringent conditions” refers to similar conditions to those that produce hybridization with DNA probes at least 40 nucleotides in length, such as a buffer containing 0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (component V) at a reaction temperature of 65°C, or a buffer containing 48% formamide, 4.8 × SSC, 0.2 M Tris-Cl, pH 7.6, 1X Denhardt solution, 10% dextran sulfate, and 0.1% SDS at a reaction temperature of 42°C. Other reaction conditions for high-strict hybridization, such as PCR, Northern, Southern or in situ hybridization, DNA sequencing, etc., are well known to technicians in the field of molecular biology (Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons, New York, NY, 1998.).

[0097] As used herein, the term "strand displacement" or its grammatical variations are terms used in the art and refer to the ability of a polymerase to displace a downstream complementary nucleic acid strand it encounters during the synthesis of a new complementary strand. The result is a double-stranded nucleic acid molecule containing both the original template strand and the newly synthesized complementary strand, while the original complementary strand is removed. Several DNA polymerases have been reported to possess varying degrees of strand displacement activity. For example, phi29 DNA polymerase exhibits a strong strand displacement ability. Other examples of strand displacement polymerases include DNA polymerase I, DNA polymerase I large fragment (Klenow), DNA polymerase, and Bacillus stearothermophilus (Bst) DNA polymerase (large fragment).

[0098] Some strand displacement polymerases are also known to be heat-resistant. For example, Bst DNA polymerase (large fragment) exhibits good strand displacement activity at high temperatures (e.g., about 65°C). Other such examples include, but are not limited to, DNA polymerase I, and DNA polymerase I large fragment (Klenow) exhibits good strand displacement activity at high temperatures (e.g., around 37°C). DNA polymerase exhibits good strand displacement activity at high temperatures (e.g., around 75°C).

[0099] Several chain displacement polymerases are commercially available. For example, New England Polymerase... Several engineered Bst DNA polymerases have been commercialized. Manufacturer instructions and recommendations for these products (available at www.neb.com / faqs / 0001 / 01 / 01 / when-should-bst-dna-polymerase-be-the-choiceme-of-choice) are provided for illustrative purposes only. Figure 9 In the middle of replication. Zeng et al. described one of the mutants of DNA polymerase I—a large fragment of DNA polymerase I (Klenow) lacking 5′→3′ exonuclease activity but retaining strand displacement activity (Klenow exo). - (Zeng et al., “Strand Displacement Amplification for Multiplex Detection of Nucleic Acids” (2018); DOI:10.5772 / intechopen.80687). Although these enzymes can be used in conjunction with the methods and kits of the present invention, the present invention is by no means limited to these exemplary commercial enzymes. It will be understood by those skilled in the art that other enzymes currently known or to be discovered in the art that satisfy the activities described in this disclosure are also covered and included in this disclosure.

[0100] The terms "subject" and "patient" are used interchangeably. As used herein, in some embodiments, the subject is a mammal, such as a nonprimate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In certain embodiments, the subject is a human.

[0101] As used herein, the term "thermostable polymerase" refers to a polymerase that is stable and active within a temperature range of 50-80°C and catalyzes the extension of a primer that binds to complementary bases of the template strand after annealing, resulting in a new strand. Synthesis can begin at the 3' end of the primer and proceed toward the 5' end of the template strand (5'→3' polymerase activity) until synthesis terminates, producing nucleic acid molecules of varying lengths. Alternatively, synthesis can begin at the 5' end of the primer and proceed toward the 3' end of the template strand (3'→5' polymerase activity). Thermostable polymerases that are inactive at lower temperatures outside the aforementioned temperature range but can be activated or reactivated when exposed to temperatures within the aforementioned temperature range are referred to herein as heat-activated enzymes. Thermostable polymerases that are inactive at higher temperatures outside the aforementioned temperature range but can be activated or reactivated when exposed to temperatures within the aforementioned temperature range are referred to herein as heat-inactivated enzymes.

[0102] 4.3 Primers

[0103] According to this disclosure, primers are designed to serve as the starting point for the synthesis of primer extension products (i.e., amplification products) when placed under suitable conditions (e.g., the presence of nucleotides and an inducing agent, such as DNA polymerase, and at suitable temperature and pH). In some embodiments, primers are preferably single-stranded to achieve maximum amplification efficiency, but may alternatively be provided in double-stranded form. In those embodiments where primers are provided in double-stranded form, the primers may be pretreated to separate their strands before being isolated. The primers are used to produce primer extension products. In some embodiments, the primers are oligodeoxyribonucleotides. In other embodiments, the primers are oligoribonucleotides.

[0104] In some embodiments, a pair of upstream and downstream primers are designed such that they operatively define an amplification region or sequence in a target nucleic acid molecule, meaning that the primers have sequences designed to specifically hybridize with the two ends of the region to be amplified in the target nucleic acid molecule. According to this disclosure, in some embodiments, the primers are designed to be substantially complementary to the template strand in the target nucleic acid, meaning that the base pairing between the primer and the target is sufficient to initiate hybridization for primer extension. The percentage of base pairings between two sequences considered “substantially complementary” also depends on the stringency of the hybridization conditions, and such a percentage and choice of conditions will be common and readily understood by those skilled in the art, given the content of this disclosure.

[0105] In some embodiments, a target sequence is selected before performing the method. Specifically, in some embodiments, the selection of the target sequence is based on determining the genus and species of the target organism. In some embodiments, a genomic sequence that is relatively abundant in the organism is selected as the target. In some embodiments, the target sequence is selected from ribosomal RNA (rRNA) coding genes or mitochondrial genes. In some embodiments, a genomic sequence unique to the relevant organism is selected. For example, to identify a unique sequence for an organism, in some embodiments, candidate sequences of the organism of interest are compared with sequences of other closely related species in evolution (e.g., orthologous genes of different species). Orthologs are one or more genes that are vertically homologous and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of epoxide hydrolysis. For example, genes are vertically homologous when they share a sufficient amount of sequence similarity to indicate that they are homologous, or are related through evolution of a common ancestor. In some embodiments, a cross-species conserved genomic sequence is selected as the target. In some embodiments, a genomic sequence that tends to have the desired genetic mutation is selected as the target.

[0106] Therefore, in some embodiments, the primer sequence is not perfectly complementary to the template strand of the target nucleic acid molecule, and the primer sequence can be optimized even if the target sequence is determined. For example, in some embodiments, a non-complementary fragment can be ligated to the 5' end of the primer, while the remainder of the primer sequence is complementary to the strand. For example, in other embodiments, the primer contains non-complementary bases or fragments scattered within regions complementary to the target. As will be appreciated by those skilled in the art, a variety of primers can be designed as long as the primer has sufficient base pairing with the template strand to be amplified to hybridize and form a template for the next round of amplification.

[0107] Without being bound by theory, it can be expected that the amplification rate of the SEA method is affected by at least the following three factors: (1) the probability of forming denatured vesicles, (2) the amplification efficiency of polymerase, and (3) the efficiency of primers in specifically binding to the target sequence in denatured vesicles.

[0108] Specifically, the formation of denaturation bubbles and the amplification efficiency of polymerase are affected by the reaction temperature. (Chander et al. "A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP)," Front. Microbiol., (2014) 5:395; Sanchez et al. "DNA kinks and bubbles: temperature dependence of the elastic energy of sharply bent 10-nm-size DNA molecules," Physical Review E (2013) 87:22710). As the temperature increases, the dynamic opening and closing of denaturation bubbles in double-stranded DNA molecules becomes more frequent (Adamcik et al., "Quantifying supercoiling-induced denaturation bubbles in DNA," Soft Matter, (2012) 8:8651-8658).

[0109] Furthermore, the amplification efficiency of polymerase is also affected by the reaction temperature. Specifically, the reaction temperature at which the enzyme activity reaches its maximum level is called the optimal temperature for that particular enzyme. For example, the optimal temperature for Bst DNA polymerase has been reported to be 65°C (Kucera et al. "DNA-dependent DNA polymerases," Current protocols in molecular biology, (2008) 84:3-5).

[0110] Finally, the efficiency of primer-target binding is affected by the relationship between reaction temperature and primer melting temperature (Tm or Tm value), and the Tm value depends on the primer sequence (e.g., G / C content). Generally, when the reaction temperature is close to the primer's Tm value, the primer will effectively bind to its target. However, compared to the primer's Tm value, excessively high reaction temperatures will hinder primer-target binding, while excessively low reaction temperatures will lead to excessive nonspecific primer binding and amplification (Kwok et al., "Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies," Nucleic Acids Res., (1990) 18: 999-1005; -Fernández in Methods in Enzymology, Elsevier, Editon edn., (2013), vol. 529, pp. 1-21). Methods for designing primers with specific Tm values ​​and methods for determining the optimal temperature for a given enzyme are known in the art. Furthermore, suitable reaction temperatures and primer Tm values ​​can be determined using methods known in the art, including but not limited to the exemplary steps described in Example 6 of this patent (Section 5.7.1).

[0111] In some embodiments, the Tm value of the primer is within ±5°C of the polymerase's optimal temperature. In some embodiments, the Tm value of the primer is within ±4°C of the polymerase's optimal temperature. In some embodiments, the Tm value of the primer is within ±3°C of the polymerase's optimal temperature. In some embodiments, the Tm value of the primer is within ±2°C of the polymerase's optimal temperature. In some embodiments, the Tm value of the primer is within ±1°C of the polymerase's optimal temperature. In some embodiments, the Tm value of the primer is within ±0.5°C of the polymerase's optimal temperature.

[0112] For example, in a specific embodiment where the polymerase is Bst DNA polymerase, the Tm value of the primers used for the reaction is selected from about 58°C to 68°C. In various embodiments where the polymerase is Bst DNA polymerase, the Tm value of the primers used for the reaction is about 58°C, about 58.5°C, about 59°C, about 59.5°C, 60°C, about 60.5°C, about 61°C, about 61.5°C, about 62°C, about 62.5°C, about 63°C, about 63.5°C, about 64°C, about 64.5°C, about 65°C, about 65.5°C, about 66°C, about 66.5°C, about 67°C, about 67.5°C, or about 68°C.

[0113] In some embodiments, the Tm values ​​of the two primers in a primer pair are approximately the same. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 5%. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 4%. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 3%. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 2%. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 1%. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 0.5%.

[0114] In some embodiments, the Tm values ​​of the two primers in a primer pair are approximately the same. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 5°C. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 4°C. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 3°C. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 2°C. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 1°C. In a particular embodiment, the Tm values ​​of a pair of primers differ from each other by less than about 0.5°C.

[0115] Unbound by theory, it is anticipated that the primers used in the method of this invention will hybridize with the target nucleic acid molecule when the target molecule is only partially denatured. Furthermore, it is anticipated that denaturation vesicles will dynamically open and close within the target nucleic acid molecule, resulting in a much shorter time window for specific primer hybridization than in conventional PCR, where the target molecule is fully denatured before primer annealing. Additionally, it is anticipated that stable hybridization between the primer and template strands within the target nucleic acid molecule will promote polymerase-catalyzed primer elongation. Further consideration is given that while GC base pairing is generally more stable, AT base pairing can hybridize at a faster rate. (Raymaekers et al., “Checklist for optimization and validation of real-time PCR assays,” J. Clin. Lab. Anal., (2009) 23:145-151).

[0116] Therefore, in some embodiments, the primers used in this method are designed to have a suitable G / C content. In specific embodiments, the primers have a suitable G / C content at the ends where the polymerase initiates primer extension. For example, in some embodiments, when the polymerase extends the primer from its 3' end, the primer can be specifically designed to have a suitable G / C content in a region closer to its 3' end, so that the primer can rapidly and stably hybridize with the template strand. Alternatively, in those embodiments where the polymerase extends the primer from its 5' end, the primer can be specifically designed to have a suitable G / C content in a region closer to its 5' end, thereby allowing the primer to rapidly form a stable hybridization with the template strand. The suitable G / C content of the primer can be determined using methods known in the art, including but not limited to the exemplary method described in Embodiment 6 of this patent (Section 5.7.2).

[0117] In certain embodiments where polymerase initiates primer extension at the 3' end of the primer, the primer uses G or C as the 3' terminal nucleotide. In some embodiments, the G / C content of the primer is from about 40% to about 60%. In certain embodiments, the G / C content of the primer is about 40%. In certain embodiments, the G / C content of the primer is about 45%. In certain embodiments, the G / C content of the primer is about 50%. In certain embodiments, the G / C content of the primer is about 55%. In certain embodiments, the G / C content of the primer is about 60%.

[0118] In certain embodiments, the primer contains at least 40% G / C content in a continuous 5-nt region including a 3' terminal nucleotide. In certain embodiments, the primer contains at least 40% G / C content in a continuous 5-nt region including a 3' terminal nucleotide, wherein the 3' terminal nucleotide is also G or C. In certain embodiments, the primer contains at least 60% G / C content in a continuous 5-nt region including a 3' terminal nucleotide. In certain embodiments, the primer contains at least 60% G / C content in a continuous 5-nt region including a 3' terminal nucleotide, wherein the 3' terminal nucleotide is also G or C. In certain embodiments, the primer contains at least 80% G / C content in a continuous 5-nt region including a 3' terminal nucleotide. In certain embodiments, the primer contains at least 80% G / C content in a continuous 5-nt region including a 3' terminal nucleotide, wherein the 3' terminal nucleotide is also G or C. In certain embodiments, the primer contains 100% G / C content in a continuous 5-nt region including a 3' terminal nucleotide. In a particular embodiment, the primer contains 100% G / C content in a continuous 5-nt region including a 3' terminal nucleotide, wherein the 3' terminal nucleotide is also G or C.

[0119] In certain embodiments where polymerase initiates primer extension at the 5' end of the primer, the primer has G or C as the 5' nucleotide. In some embodiments, the G / C content of the primer is from about 40% to about 60%. In certain embodiments, the G / C content of the primer is about 40%. In certain embodiments, the G / C content of the primer is about 45%. In certain embodiments, the G / C content of the primer is about 50%. In certain embodiments, the G / C content of the primer is about 55%. In certain embodiments, the G / C content of the primer is about 60%.

[0120] In certain embodiments, the primer contains at least 40% G / C content in a continuous 5-nt region including a 5' terminal nucleotide. In certain embodiments, the primer contains at least 40% G / C content in a continuous 5-nt region including a 5' terminal nucleotide, wherein the 5' terminal nucleotide is also G or C. In certain embodiments, the primer contains at least 60% G / C content in a continuous 5-nt region including a 5' terminal nucleotide. In certain embodiments, the primer contains at least 60% G / C content in a continuous 5-nt region including a 5' terminal nucleotide, wherein the 5' terminal nucleotide is also G or C. In certain embodiments, the primer contains at least 80% G / C content in a continuous 5-nt region including a 5' terminal nucleotide. In certain embodiments, the primer contains at least 80% G / C content in a continuous 5-nt region including a 5' terminal nucleotide, wherein the 5' terminal nucleotide is also G or C. In certain embodiments, the primer contains 100% G / C content in a continuous 5-nucleotide region including a 5' terminal nucleotide. In a particular embodiment, the primer contains 100% G / C content in a continuous 5-nt region including a 5' terminal nucleotide, wherein the 5' terminal nucleotide is also G or C.

[0121] Without being bound by theory, it is conceivable that primers with sequences capable of forming their own complementary secondary structures, or a pair of primers with sequences complementary to each other, would hinder the amplification reaction (Meagher et al., “Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA,” Analyst, (2018) 143: 1924-1933). Therefore, in some embodiments, after selecting the target sequence for primer hybridization, the primer sequences can be further optimized to avoid or reduce the likelihood of forming complementary structures within or between these primers. Methods for primer sequence optimization are known in the art, including but not limited to the exemplary methods described in Example 6 of this patent (Section 5.7.3).

[0122] Based on current findings, primer length selection can depend on a variety of factors, including but not limited to amplification reaction temperature and time. Without being theoretically limited, it can be expected that higher reaction temperatures result in longer complementary regions between the primers and the target, which can help avoid nonspecific amplification.

[0123] Beyond theoretical constraints, it can also be anticipated that reducing primer extension time in each amplification cycle can significantly reduce the total time required to produce a detectable amount of amplified product, thereby reducing the time required for target detection and related diagnostics. Therefore, in some embodiments, primers are designed to specifically hybridize with most of the amplification region, resulting in a relatively small number of nucleotides to be extended in each amplification cycle (i.e., the length difference between the amplified product and the primer).

[0124] For example, in certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 30% to about 60%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 30%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 35%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 40%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 45%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 50%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 55%. In certain embodiments, the ratio of primer length to the total length of the amplification product is in the range of about 60%.

[0125] In some embodiments, a pair of primers is designed for a relatively short amplification region, the primers having unique sequences that indicate the characteristics, state, origin, or source of the target nucleic acid. The selection of the amplification region in the target nucleic acid depends on the detection purpose or application scenario, and those skilled in the art will recognize this selection approach after reading this patent. For example, to detect the presence of gene mutations or polymorphisms in a sample, an amplification region including the expected site of the mutation or polymorphism can be selected. To detect the presence of pathological microorganisms in a sample, an amplification region covering known characteristic sequences in the microbial genome can be selected.

[0126] In some embodiments, the primer pair is designed to amplify a region less than 100 bp in length in the target nucleic acid molecule. In some embodiments, the length of the amplified fragment generated by this method is less than 90 bp. In some embodiments, the length of the amplified fragment generated by this method is less than 80 bp. In some embodiments, the length of the amplified fragment generated by this method is less than 70 bp. In some embodiments, the length of the amplified fragment generated by this method is less than 60 bp. In some embodiments, the length of the amplified fragment generated by this method is less than 50 bp. In some embodiments, the amplified fragment generated by this method is approximately 20-50 bp in length. In some embodiments, the amplified fragment generated by this method is approximately 30-50 bp in length. In some embodiments, the amplified fragment generated by this method is approximately 35-50 bp in length.

[0127] In some embodiments, to reduce the time required for primer extension, the primer pair is configured to produce a short amplification fragment of about 20 base pairs (bp) to about 50 bp in length. The amplification fragment contains at least a central portion corresponding to a unique sequence in the target nucleic acid molecule, the flanks of which may be primer sequences that are the same as or different from the sequence in the target molecule. For example, in a particular embodiment, the amplification fragment is about 20 bp in length. In a particular embodiment, the amplification fragment is about 21 bp in length. In a particular embodiment, the amplification fragment is about 22 bp in length. In a particular embodiment, the amplification fragment is about 23 bp in length. In a particular embodiment, the amplification fragment is about 24 bp in length. In a particular embodiment, the amplification fragment is about 25 bp in length. In a particular embodiment, the amplification fragment is about 26 bp in length. In a particular embodiment, the amplification fragment is about 27 bp in length. In a particular embodiment, the amplification fragment is about 28 bp in length. In a particular embodiment, the amplification fragment is about 29 bp in length. In a particular embodiment, the amplification fragment is about 30 bp in length. In a particular embodiment, the amplification fragment is about 31 bp in length. In a specific embodiment, the length of the amplified fragment is approximately 32 bp. In a specific embodiment, the length of the amplified fragment is approximately 33 bp. In a specific embodiment, the length of the amplified fragment is approximately 34 bp. In a specific embodiment, the length of the amplified fragment is approximately 35 bp. In a specific embodiment, the length of the amplified fragment is approximately 36 bp. In a specific embodiment, the length of the amplified fragment is approximately 37 bp. In a specific embodiment, the length of the amplified fragment is approximately 38 bp. In a specific embodiment, the length of the amplified fragment is approximately 39 bp. In a specific embodiment, the length of the amplified fragment is approximately 40 bp. In a specific embodiment, the length of the amplified fragment is approximately 41 bp. In a specific embodiment, the length of the amplified fragment is approximately 42 bp. In a specific embodiment, the length of the amplified fragment is approximately 43 bp. In a specific embodiment, the length of the amplified fragment is approximately 44 bp. In a specific embodiment, the length of the amplified fragment is approximately 45 bp. In a specific embodiment, the length of the amplified fragment is approximately 46 bp. In a specific embodiment, the length of the amplified fragment is approximately 47 bp. In a specific embodiment, the length of the amplified fragment is approximately 48 bp. In a specific embodiment, the length of the amplified fragment is approximately 49 bp. In a particular embodiment, the length of the amplified fragment is approximately 50 bp.

[0128] In some embodiments, to reduce the time required for primer extension, the primers are configured to specifically hybridize with a large portion of the amplified region in the target molecule. Specifically, in some embodiments where the amplified product is about 20 to 50 bp in length, at least one of the primers in a pair is about 10 to 25 nucleotides (nt) in length. In some embodiments, where the amplified product is about 20 to 50 bp in length, both primers in the primer pair are about 10 to 25 nt in length.

[0129] Specifically, in some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 10 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 11 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 12 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 13 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 14 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 15 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 16 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 17 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 18 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 19 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 20 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 21 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 22 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 23 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 24 nt in length. In some embodiments where the amplification product is approximately 20 to 50 bp in length, at least one of the primers in a pair is approximately 25 nt in length.

[0130] As those skilled in the art will understand, in actual primer design, the selection of primer sequences based on different considerations (e.g., Tm, G / C content, sequence complementarity) may be contradictory. Therefore, in some embodiments, different considerations can be compared with each other to determine the priority order among the different considerations. In particular, when the selection of primer sequences based on lower priority considerations contradicts the selection of primer sequences based on higher priority considerations, the selection based on higher priority considerations can be adopted. Example 6 further provides an example procedure for determining such a priority order as in Section 5.7.4.

[0131] 4.4 Enzymes

[0132] According to this disclosure, the polymerase that can be used in conjunction with this method includes a thermostable polymerase having chain displacement activity in a temperature range of about 50-80°C. In some embodiments, the thermostable polymerase selected for use in the method of the present invention has chain displacement activity at a temperature of about 70-80°C. In some embodiments, the thermostable polymerase has 5′→3′ polymerase activity and is capable of extending the primer bound to the template strand after annealing from the 3′ end to the 5′ end of the template strand, thereby displacing the original complementary strand along the 5′→3′ direction. In other embodiments, the thermostable polymerase has 3′→5′ polymerase activity and is capable of extending the primer bound to the template strand after annealing from the 5′ end to the 3′ end of the template strand, thereby displacing the original complementary strand along the 3′→5′ direction.

[0133] In contrast to strand displacement, some polymerases (e.g., Taq DNA polymerase) degrade the downstream complementary strand encountered via exonuclease activity. Although the result is also the formation of a double strand with the original template strand and a newly synthesized complementary strand (removed by degradation), the exonuclease activity can reduce the total amount of amplified nucleic acid fragments, thus making it less than ideal in some (but not all) applications. Therefore, some polymerases have been engineered to remove the 5′→3′ exonuclease activity of the wild-type enzyme while retaining both polymerase and strand displacement activities. Thus, in some embodiments, the thermostable polymerase has 5′→3′ polymerase activity but no 5′→3′ exonuclease activity. In some embodiments, the thermostable polymerase has 3′→5′ polymerase activity but no 3′→5′ exonuclease activity.

[0134] In some embodiments, the thermostable polymerase is a heat-activated enzyme. In some embodiments, the thermostable polymerase is a heat-inactivated enzyme. In some embodiments, the thermostable polymerase has reverse transcriptase activity. In some embodiments, the thermostable polymerase has an amplification rate of at least 10 nt / s at its optimal temperature.

[0135] Examples of thermostable polymerases that may be used in conjunction with this patent include, but are not limited to, phi29 DNA polymerase or a truncated or mutated form thereof, DNA polymerase I or a truncated or mutated form thereof, DNA polymerase or a truncated or mutated form thereof, and Bacillus stearothermophilus (Bst) DNA polymerase or a truncated or mutated form thereof, and Taq aquatic thermophilus (Taq) DNA polymerase or a truncated or mutated form thereof.

[0136] In some embodiments, the polymerase is a Bst DNA polymerase. In some embodiments, the polymerase is a full-length Bst DNA polymerase. In some embodiments, the polymerase is a Bst DNA polymerase (large fragment). In some embodiments, the polymerase is a mutant form of the Bst DNA polymerase. In a particular embodiment, the mutant Bst DNA polymerase lacks 5′→3′ exonuclease activity. In some embodiments, the Bst DNA polymerase is commercially available. In a particular embodiment, the Bst DNA polymerase is selected from New England... Commercially available Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, and Bst 3.0 DNA polymerase.

[0137] In some embodiments, the polymerase is DNA polymerase I or a mutant or truncated form thereof. In some embodiments, the polymerase is a large fragment of wild-type DNA polymerase I (Klenow). In some embodiments, the polymerase is Klenow exo - In some embodiments, the polymerase is phi29 DNA polymerase or a mutant or truncated form thereof. In some embodiments, the polymerase is... DNA polymerase, Deep (exo - DNA polymerase, Deep DNA polymerase, or (exo - DNA polymerase.

[0138] In some embodiments, the polymerase is Taq DNA polymerase. In some embodiments, the polymerase is a mutant form of Taq DNA polymerase. In some embodiments, the Taq DNA polymerase is a heat-activated enzyme. In some embodiments, the Taq DNA polymerase is commercially available. In a specific embodiment, the Taq DNA polymerase is selected from those available from New England... Purchase hot-start Taq DNA polymerase. Hot-start Taq DNA polymerase, OneTaq DNA polymerase, hot start DNA polymerase and Taq DNA polymerase. In some embodiments, the Taq DNA polymerase is LongTaq DNA polymerase.

[0139] While the exemplary enzymes or corresponding commercial products described above can be used in conjunction with the methods and kits of this invention, the selection of enzymes in this invention is by no means limited to those listed above. As will be understood by those skilled in the art, other enzymes currently known or to be discovered in the future that satisfy this patent are also considered and included in this patent. Other polymerases suitable for the content of this patent can be generated and selected using methods known in the art. For example, wild-type polymerases can be mutated by directed or random mutagenesis to produce peptide variants, which can then be screened (e.g., alone or by high-throughput assays) to identify mutants with the desired polymerase activity. For illustrative purposes, several exemplary methods for enzyme mutagenesis and evolution are provided below.

[0140] Directed evolution is a powerful method that involves introducing mutations targeting a specific gene or an oligonucleotide sequence containing that gene to improve and / or alter an enzyme, protein, or peptide (e.g., polymerase, particularly DNA polymerase). Modified and / or altered enzymes, proteins, or peptides can be identified by developing and implementing sensitive, high-throughput detection methods that can automatically screen multiple enzyme or peptide variants (e.g., >1.0 × 10⁻⁶). 4Iterative mutagenesis and screening are typically performed to provide enzymes or peptides with optimized properties. Computational algorithms have also been developed to help identify mutagenic gene regions and can significantly reduce the number of enzyme or peptide variants that need to be generated and screened (Fox, RJ, et al., Trends Biotechnol., 2008, 26, 132-138; Fox, RJ, et al., Nature Biotechnol., 2007, 25, 338-344). Many directed evolution techniques have been developed and proven to be effective in creating various mutant libraries. These methods have been successfully applied to improve a variety of properties of many enzymes and proteins (Hibbert et al., Biomol. Eng., 2005, 22, 11-19; Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries, pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax, Biomol. Eng., 2005, 22, 1-9; and Sen et al., Appl. Biochem. Biotechnol., 2007, 143, 212-223). Enzyme and protein properties improved and / or altered through directed evolution include, for example: heat resistance for high-temperature reactions; pH stability for bioprocessing under low or high pH conditions; tolerance to enable substrates or products with higher activity; binding force (Km), including expanding ligand or substrate binding, including non-natural substrates; inhibition of substrates or key intermediates (Ki) to eliminate products; activity (kcat) to increase the rate of enzymatic reactions to obtain the desired changes; isoelectric point (pI) to increase the solubility of proteins or peptides; and acidity coefficient (pKa) to alter the ionization state of proteins or peptides relative to pH.

[0141] Numerous exemplary methods have been developed for the mutagenesis and diversification of genes and oligonucleotides to introduce desired properties into specific enzymes, proteins, and peptides. Such methods are well known to those skilled in the art. Any of these methods can be used to alter and / or optimize the activity of enzymes, proteins, or peptides, including polymerases such as DNA polymerases. Such methods include, but are not limited to, error-prone polymerase chain reaction (epPCR), which introduces random point mutations by reducing the fidelity of DNA polymerase in the PCR reaction (Pritchard et al., J. Theor. Biol., 2005, 234:497-509); error-prone rolling circle amplification (epRCA) is similar to epPCR, except that epRCA uses an intact circular plasmid as a template and uses a random hexamer amplified plasmid with exonuclease-resistant phosphate-thioester bonds on the last two nucleotides, which is then converted into cells that recirculate the plasmid in a tandem repeat manner (Fujii et al., Nucleic Acids Res., 2004, 32:e145; and Fujii et al., Nat. Protoc., 2006, 1, 2493-2497). DNA, gene, or family shuffling typically involves digesting two or more variant genes with nucleases (such as Dnase I or EndoV) to generate a library of random fragments, which are then reassembled through annealing and extension cycles by DNA polymerases to produce a chimeric gene library (Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91, 10747-10751; and Stemmer, Nature, 1994, 370, 389-391). Staggered expansion (StEP) requires template initiation followed by repeated two-step PCR cycles with denaturation and very short annealing / extension times (as short as 5 seconds) (Zhao et al., Nat. Biotechnol., 1998, 16, 258-261); random primer recombination (RPR) uses random sequence primers to generate numerous short DNA fragments complementary to the template fragment (Shao et al., Nucleic Acids Res., 1998, 26, 681-683).

[0142] Other methods include heteroduplex recombination, in which linearized plasmid DNA is used to form mismatch-repaired heteroduplexes (See: Volkov et al., Nucleic Acids Res., 1999, 27:e18; Volkov et al., MethodsEnzymol., 2000, 328, 456-463); and random chimerism on transient templates (RACHITT), which uses DNase I fragmentation and size grading of single-stranded DNA (ssDNA) (See: Coco et al., Nat. Biotechnol., 2001, 19, 354-359).Recombination extension with truncated templates (RETT) requires template conversion from primers for unidirectional growth in the presence of a unidirectional ssDNA fragment used as a template pool (See: Lee et al., J. Mol. Cat., 2003, 26, 119-129); Degenerate oligonucleotide gene shuffling (DOGS), where degenerate primers are used to control recombination between molecules (Bergquist and Gibbs, Methods Mol. Biol., 2007, 352, 191-204; Bergquist et al., Biomol. Eng., 2005, 22, 63-72; Gibbs et al., Gene, 2001, 271, 13-20); Incremental truncation for creating hybrid enzymes (ITCHY), which creates a combinatorial library containing a single-base-pair deletion of a gene or gene fragment of interest (See: Ostermeier et al.). al., Proc. Natl. Acad. Sci. USA, 1999, 96, 3562-3567; and Ostermeier et al., Nat. Biotechnol., 1999, 17, 1205-1209); Thiozyme truncation to produce hybrid enzymes (THIO-ITCHY), similar to ITCHY, but different in that it uses phosphate thioester dNTPs to produce truncation (See: Lutz et al., Nucleic Acids Res., 2001, 29, E16); SCRATCHY, which combines two recombinant gene methods: ITCHY and DNA shuffling (See: Lutz et al.). al., Proc. Natl. Acad. Sci. USA, 2001, 98, 11248-11253); Random drift mutagenesis (RNDM), in which mutations generated by epPCR are screened / selected to retain useful activity (See: Bergquistet al., Biomol. Eng., 2005, 22, 63-72); Sequence saturation mutagenesis (SeSaM), a random mutagenesis method that uses the random incorporation and cleavage of phosphate thioester nucleotides to generate a library of random-length fragments, which are used as templates for expansion in the presence of "universal" bases (such as inosine). The replication of inosine-containing complement produces random base incorporation and induces mutagenesis (See: Wong et al., Biotechnol. J., 2008, 3, 74-82; Wong et al., Nucleic Acids Res., 2004, 32, e26; Wong et al. al., Anal. Biochem., 2005, 341, 187-189.).Synthetic shuffling uses overlapping oligonucleotides designed to encode “all genetic diversity in the target” and allows shuffling progeny to have very high diversity (See: Nesset et al., Nat. Biotechnol., 2002, 20, 1251-1255); NexT, a nucleotide exchange and excision technology, utilizes dUTP incorporation followed by terminal DNA fragmentation with uracil-DNA glycosylase and piperidine treatment (See: Muller et al., Nucleic Acids Res., 33:e117).

[0143] Further mutagenesis methods include sequence-homology-independent protein recombination (SHIPREC), in which adapters are used to promote the fusion between two distantly related or unrelated genes, and a series of chimeras are generated between the two genes, forming single-cross hybrids (See: Sieber et al., Nat. Biotechnol., 2001, 19, 456-460); and locus saturation mutagenesis. TM (GSSM TMThe starting material includes a supercoiled double-stranded DNA (dsDNA) plasmid containing an insert fragment and two primers that denature at the desired mutation site, thereby enabling the individual introduction of all amino acid variations at each position of a protein or peptide (See: Kretz et al., Methods Enzymol., 2004, 388, 3-11); and combinatorial cassette mutagenesis (CCM), which involves replacing a specific region containing a large number of possible amino acid sequence changes with a short oligonucleotide cassette (See: Reidhaar-Olson et al. Methods Enzymol., 1991, 208, 564-586; Reidhaar-Olson et al. Science, 1988, 241, 53-57). Combinatorial multibox mutagenesis (CMCM) is essentially similar to CCM, using epPCR to identify hotspots and hotspot regions with high mutation rates, and then expanding through CMCM to cover the defined regions of the protein sequence space (See: Reetz et al., Angew. Chem. Int. Ed Engl., 2001, 40, 3589-3591); mutant technology, in which conditional ts mutant plasmids utilize the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, allows random and natural mutation frequencies to increase by 20 to 4000 times during selection and prevents the accumulation of harmful mutations when selection is not needed (See: Selifonova et al., Appl. Environ. Microbiol., 2001, 67, 3645-3649; Low et al., J. Mol. Biol., 1996, 260, 3659-3680).

[0144] Other exemplary methods include look-through mutagenesis (LTM), a multidimensional mutagenesis method used to evaluate and optimize mutations in selected amino acid combinations (See: Rajpal et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 8466-8471.); and gene recombination, a homology-independent DNA rearrangement method that can be applied to multiple genes at once or to create large chimeric (multiple mutation) libraries of single genes (See: Short, JM, US Patent 5,965,408, Tunable Gene Reassembly). TM); Silico protein design automation (PDA) is an optimization algorithm that anchors the protein backbone with a specific folding structure and searches the sequence space for amino acid substitutions that stabilize protein folding and overall protein energy. It is generally most efficient for proteins with known three-dimensional structures (See: Hayes et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15926-15931); Iterative saturation mutagenesis (ISM) involves using structure / function knowledge to select possible enzymatic modification sites. Mutagenesis methods (e.g., Stratagene QuikChange (Stratagene; San Diego CA)) are used to saturate the selected sites, screen / select for desired properties, and use modified clones to start from another site and repeat until the desired activity is obtained (See: Reetz et al., Nat. Protoc., 2007, 2, 891-903; Reetz et al., Angew. Chem. Int. Ed. Engl., 2006, 45, 7745-7751.

[0145] In addition to the biological methods described above, the evolution of enzymes (e.g., polymerases) can also be carried out using chemical synthesis methods. For example, large combinatorial peptide libraries (e.g., >1.0 × 10⁻⁶) containing mutants can be synthesized using known solution-phase or solid-phase peptide synthesis techniques. 6 (See review: Shin, D.-S., et al., J. Biochem. Mol. Bio., 2005, 38, 517-525.). Chemical peptide synthesis methods can be used to produce polymerase variants containing a variety of α-amino acids, including native proteogenic amino acids, as well as non-native and / or non-proteogenic amino acids, such as amino acids with non-proteogenic side chains, or D-amino acids, or β-amino acids.

[0146] Any of the above-described methods for enzyme mutagenesis can be used alone or in any combination to improve the performance of enzymes, proteins, and peptides. Similarly, any of the above-described mutagenesis methods and / or the contents shown can be used alone or in any combination to enable the production of polymerase variants, which can be selected to improve properties.

[0147] In some embodiments, the mutant polymerase has at least 80% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 85% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 90% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 95% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 96% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 97% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 98% of its nucleic acid sequence identical to the corresponding wild-type polymerase. In some embodiments, the mutant polymerase has at least 99% of its nucleic acid sequence identical to the corresponding wild-type polymerase.

[0148] Methods for determining sequence identity are known in this field. For example, examining the nucleic acid or amino acid sequences of two polypeptides will reveal the identity and similarity between the compared sequences. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W, and others, can be used to compare and determine the similarity or identity of original sequences, and can also determine the presence or importance of vacancies in sequences that can be assigned weights or scored. Such algorithms are also known in the art and are similarly applicable to determining the similarity or identity of nucleotide sequences. Based on well-known methods for calculating statistical similarity, or the chance of finding similar matches in random polypeptides and determining the significance of the matches, parameters sufficient to determine similarity are calculated. If desired, two or more sequences can be compared using a computer by those skilled in the art for visual optimization.

[0149] For example, exemplary parameters for determining the correlation of two or more sequences using the BLAST algorithm are shown below. In short, amino acid sequence alignment can be performed using BLASTP version 2.0.8 (Jan-05-1999) with the following parameters: matrix: 0BLOSUM62; vacancy open: 11; gap extension: 1; x_dropoff: 50; expectation: 10.0; word count: 3; filter: on. Nucleic acid sequence alignment can be performed using BLASTN version 2.0.6 (September 16, 1998) with the following parameters: match: 1; mismatch: -2; vacancy open: 5; gap extension: 2; x_dropoff: 50; expectation: 10.0; word count: 11; filter: off. Those skilled in the art will understand how the above parameters can be modified, for example, to increase or decrease the stringency of the comparison, and to determine the correlation of two or more sequences.

[0150] In some embodiments, the functional variant of the protein comprises one or more conserved substitutions compared to the wild-type counterpart. In some embodiments, the functional variant of the protein comprises one or more amino acid residues substituted with non-natural amino acid residues compared to the wild-type counterpart.

[0151] Wild-type and mutant enzymes (e.g., polymerases) can be screened to select enzymes with the properties desired by the method of the present invention for use in conjunction with the method. In some embodiments, screening is performed on enzymes and / or mutant variants that retain at least DNA polymerase activity and strand substitution activity. In some embodiments, screening is performed on enzymes and / or mutant variants that have thermostability and activity in a temperature range of about 50-80°C. In some embodiments, screening is performed on enzymes and / or mutant variants that have thermostability and activity in a temperature range of about 70-80°C. In some embodiments, screening is performed on enzymes and / or mutant variants that have an optimal temperature in a temperature range of 50-80°C. In some embodiments, screening is performed on enzymes and / or mutant variants that have an optimal temperature in a temperature range of 70-80°C. In some embodiments, screening is performed on enzymes and / or mutant variants that have an extension rate of at least 10 nt / s at the optimal temperature of 50-80°C. In some embodiments, screening is performed on enzymes and / or mutant variants that have reverse transcriptase activity. In some embodiments, screening is performed on enzymes and / or mutant variants that do not have exonuclease activity. In some embodiments, heat-activated and / or heat-inactivated enzymes are screened against enzyme and / or mutant variants.

[0152] Screening can be performed using methods and assays known in the art. For example, whether a given polymerase has chain displacement activity can be determined using chain displacement amplification (SDA) assays described or used by Walker et al. (Nucleic Acids Res. 1992 Apr 11; 20(7): 1691–1696) and Gao et al. (Nucleic Acids Res., 2009 Feb 1; 37, e20.). Thermodynamic properties of a given enzyme, including its optimal temperature and extension rate, can be determined using assays such as those described by Rychlik et al. (Nucleic Acids Res., 1990 Nov 21; 18(21), 6409–6412.). Whether a polymerase possesses reverse transcriptase activity can be determined using the assays described or used by Shi et al. (J. Am. Chem. Soc., 2015 Oct 16; 137(43), 13804-13806) and Lanford et al. (J. Virol., 1995 Apr 21; 69(7), 4431-4439.). Whether a polymerase possesses exonuclease activity can be determined using the assays described or used by Holland et al. (P. Natl. Acad. Sci. USA, 1991 Aug 15; 88(16), 7276-7280) and Beese et al. (Beese et al., EMBO J., 1991 Jan 1; 10(1), 25-33.).

[0153] Methods for rapidly screening large numbers of different mutant enzymes, proteins, or peptide variants involve the use of display technologies (For areview, see: Ullman, CG, et al., Briefings Functional Genomics, 2011, 10, 125-134). Peptide display technologies offer the advantages that specific peptide-coding information (e.g., RNA or DNA sequence information) can be linked to or otherwise associated with each corresponding peptide in the library, and this information is accessible and readable after the screening event (e.g., by amplification and sequencing), enabling the identification of individual peptides in a large library that exhibit desirable properties (e.g., high binding affinity). Enzyme peptide mutants exhibiting the desired improved properties (hits) can be further mutagenized to produce highly optimized enzyme variants.

[0154] 4.5 Methods

[0155] One currently available approach provides a method for amplifying and detecting target nucleic acids in samples. This approach significantly improves upon existing denaturation bubble-mediated strand exchange amplification (SEA) techniques, which were first reported by Shi et al. in 2016 (Shi et al. “Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification” Chem Commun (Camb) (2016) 4; 52(77): 11551-4).

[0156] Shi et al. reported a SEA analysis for exponential DNA amplification under isothermal conditions using Bst DNA polymerase and a pair of specific primers. The isothermal SEA method relies on the spontaneous formation of denatured regions (“denatured vesicles”) within double-stranded DNA (dsDNA) due to environmental thermal fluctuations. A pair of oligonucleotide primers then penetrates the denatured vesicles, binding to the unentangled single-stranded DNA within, which, under the action of the polymerase, extends and replaces the original complementary strand to generate the amplified fragment. Therefore, this method is considered to take advantage of the spontaneous formation of small denatured vesicles without heating the sample, thus advantageously eliminating the need for a thermal cycler and allowing PCR reactions to be performed at temperatures where the polymerase typically exhibits optimal activity (Shi et al., 2016, ibid.).

[0157] Since its establishment, the isothermal SEA method has been proven and used for the rapid detection and diagnosis of various pathogens, such as Listeria monocytogenes (Zhang et al. “Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification,” Analytical Biochemistry, (2018) 545:38-42); Mycoplasma pneumoniae (Shi et al. “Rapid diagnosis of Mycoplasma pneumoniae infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR,” Scientific Reports, vol. 9; article number: 896 (2019)); Staphylococcus aureus (Liu et al., “Rapid and Simple Detection of Viable Foodborne Pathogen Staphylococcus aureus,” Front Chem. (2019) Mar 12; 7:124); Escherichia coli (Chinese Patent Application Publication No.: CN 105176971A); and pine wilt nematode (Liu et al.). (e.g., “The Rapid detection of the Bursaphelenchus Xylophilus by Denaturation Bubble-mediated Strand Exchange Amplification,” Anal. Sci. 2019, 18P-461P.”); and adulterated meat (Liu et al., “A simple isothermal nucleic acid amplification method for the effective on-site identification for adulteration of pork source inmutton,” Food Control, 2019, 98 297-302). The isothermal SEA method has also been demonstrated to detect trace amounts of target nucleic acids in samples (concentrations as low as 1.0 × 10⁻⁶). -14The ability of Bst DNA polymerase to amplify and detect target RNA molecules in samples has been demonstrated (Chinese Patent Application Publication No.: CN 109136337A). Furthermore, Bst DNA polymerase has been found to possess inherent reverse transcriptase activity (Shi et al. "Innate reverse transcriptase activity of DNA polymerase for isothermal RNA direct detection." J. Am. Chem. Soc. (2015) 137, 13804–13806). It has been confirmed that isothermal SEA reactions using Bst DNA polymerase can effectively amplify and detect target RNA molecules in samples without the need for another reverse transcriptase (Chinese Patent Application Publication No.: CN 105176971 A).

[0158] The surprising finding of this disclosure is that even within a small range of a few degrees Celsius, rapidly altering the reaction temperature to change the isothermal SEA method significantly improves amplification efficiency and rate by thousands of times. Therefore, in certain paragraphs of this application, this method is referred to as "accelerated SEA." Not bound by theory, this disclosure envisions promoting denaturing vesicle formation by inducing temperature fluctuations within a small temperature range, which makes it more efficient for primer entry and hybridization. Furthermore, since the temperature fluctuations are near the optimal temperature for polymerase-catalyzed primer extension, the increased frequency of denaturing vesicle occurrence does not come at the expense of an increased extension rate. In addition to the small range of temperature fluctuations, the amplified fragments of this reaction are very short (typically 20-50 bp). Therefore, even if the polymerase activity may be slightly reduced, the polymerase can still achieve the amplification of such short fragments. Thus, in various examples, the induced temperature fluctuations are within 1°C to 15°C of the polymerase's optimal extension temperature. In various embodiments, the temperature fluctuation range used in this method is approximately less than 30°C. In various embodiments, the temperature fluctuation range used in this method is approximately less than 25°C. In various embodiments, the temperature fluctuation range used in this method is approximately less than 20°C.

[0159] In some embodiments, the method includes contacting a polymerase and a pair of specific oligonucleotide primers with a sample containing or suspected of containing the target nucleic acid to form an amplification mixture. The method also includes subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify the sequence of the target nucleic acid via polymerase chain reaction (PCR). The generation of the amplified fragment can then be detected, and this detection can be used as the basis for various analyses and diagnostics.

[0160] According to this disclosure, at least one of the first and second temperatures is suitable for (a) forming denaturing vesicles in a double-stranded target molecule; (b) primers specifically hybridizing with the target nucleic acid; (c) polymerase-catalyzed amplification of primer extension in the mixture; or any combination of (a) to (c). In some embodiments, a temperature range between the first and second temperatures is suitable for (a) forming denaturing vesicles in a double-stranded target molecule; (b) primers specifically hybridizing with the target nucleic acid; (c) polymerase-catalyzed amplification of primer extension in the mixture; or any combination of (a) to (c).

[0161] In some embodiments, the selection of the first or second temperature is based on the type of polymerase used for amplification. In some embodiments, the second temperature is selected near the optimal temperature of the polymerase used. Methods for determining the optimal temperature of the enzyme are known in the art. For example, to determine the optimal temperature at which a given polymerase catalyzes primer extension under given conditions, an amplification mixture of multiple aliquots can be prepared, each aliquot containing the target polymerase, the same primers, the target, and the same concentrations of other reactants. The aliquots can be subjected to PCR at different temperature conditions, and the optimal temperature can be determined by comparing amplification rates (e.g., using real-time PCR monitoring). Furthermore, the optimal extension temperature of the polymerase can be determined based on reports in the art or recommendations from commercial polymerase manufacturers.

[0162] In some embodiments, the second temperature is selected from the polymerase optimum temperature ±6°C. For illustrative and example purposes only, if the polymerase optimum extension temperature is 65°C, then in some embodiments, the second temperature may be selected from about 59-71°C. Specifically, in this example, the second temperature may be about 59°C, about 59.5°C, about 60°C, about 60.5°C, about 61°C, about 61.5°C, about 62°C, about 62.5°C, about 63°C, about 63.5°C, about 64°C, about 64.5°C, about 65°C, about 65.5°C, about 66°C, about 66.5°C, about 67°C, about 67.5°C, about 68°C, about 68.5°C, about 69°C, about 69.5°C, about 70°C, about 70.5°C, or about 71°C. In other embodiments, the second temperature is selected from the polymerase optimum extension temperature ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0163] In various embodiments, the first temperature is about 1°C higher or lower than the second temperature, up to about 30°C. In various embodiments, the first temperature is about 1°C higher or lower than the second temperature, up to about 25°C. In various embodiments, the first temperature is about 1°C higher or lower than the second temperature, up to about 20°C. In some embodiments, the first temperature is about 1°C higher or lower than the second temperature. In some embodiments, the first temperature is about 1.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 2°C higher or lower than the second temperature. In some embodiments, the first temperature is about 2.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 3°C ​​higher or lower than the second temperature. In some embodiments, the first temperature is about 3.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 4°C higher or lower than the second temperature. In some embodiments, the first temperature is about 4.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 5.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 6°C higher or lower than the second temperature. In some embodiments, the first temperature is about 6.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 7°C higher or lower than the second temperature. In some embodiments, the first temperature is about 7.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 8°C higher or lower than the second temperature. In some embodiments, the first temperature is about 8.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 9°C higher or lower than the second temperature. In some embodiments, the first temperature is about 9.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 10°C higher or lower than the second temperature. In some embodiments, the first temperature is about 10.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 11°C higher or lower than the second temperature. In some embodiments, the first temperature is about 11.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 12°C higher or lower than the second temperature. In some embodiments, the first temperature is about 12.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 13°C higher or lower than the second temperature. In some embodiments, the first temperature is about 13.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 14°C higher or lower than the second temperature. In some embodiments, the first temperature is about 14.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 15°C higher or lower than the second temperature. In some embodiments, the first temperature is about 15.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 16°C higher or lower than the second temperature. In some embodiments, the first temperature is about 16.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 17°C higher or lower than the second temperature. In some embodiments, the first temperature is about 17.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 18°C ​​higher or lower than the second temperature.In some embodiments, the first temperature is about 18.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 19°C higher or lower than the second temperature. In some embodiments, the first temperature is about 19.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 20°C higher or lower than the second temperature. In some embodiments, the first temperature is about 20.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 21°C higher or lower than the second temperature. In some embodiments, the first temperature is about 21.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 22°C higher or lower than the second temperature. In some embodiments, the first temperature is about 22.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 23°C higher or lower than the second temperature. In some embodiments, the first temperature is about 23.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 24°C higher or lower than the second temperature. In some embodiments, the first temperature is about 24.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 25°C higher or lower than the second temperature. In some embodiments, the first temperature is about 25.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 26°C higher or lower than the second temperature. In some embodiments, the first temperature is about 26.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 27°C higher or lower than the second temperature. In some embodiments, the first temperature is about 27.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 28°C higher or lower than the second temperature. In some embodiments, the first temperature is about 28.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 29°C higher or lower than the second temperature. In some embodiments, the first temperature is about 29.5°C higher or lower than the second temperature. In some embodiments, the first temperature is about 30°C higher or lower than the second temperature.

[0164] In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 90°C. In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 89°C. In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 88°C. In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 87°C. In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 86°C. In some embodiments, the first temperature is no more than about 25°C higher than the second temperature, and the first temperature is equal to or less than about 85°C. In any of the embodiments described in this paragraph, the polymerase may be Bst DNA polymerase, Taq DNA polymerase, or DNA polymerase I. DNA polymerase, phi29 DNA polymerase, or any truncated or mutant form of these polymerases.

[0165] In some embodiments, the polymerase is a Bst DNA polymerase, and the first temperature is selected from the range of about 68-78°C, and the second temperature is selected from the range of about 55-69°C. Specifically, in a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 68°C, and the second temperature is selected from the range of about 55-69°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 68.5°C, and the second temperature is selected from the range of about 55-69°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 69°C, and the second temperature is selected from the range of about 55-69°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 69.5°C, and the second temperature is selected from the range of about 55-69°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 70°C, and the second temperature is selected from the range of about 55-69°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 70.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 71°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 71.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 72°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 72.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 73°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 73.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 74°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 74.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 75°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 75.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 76°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 76.5°C, and the second temperature is selected from the range of about 55-69°C.In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 77°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 77.5°C, and the second temperature is selected from the range of about 55-69°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is about 78°C, and the second temperature is selected from the range of about 55-69°C. Specifically, in any embodiment described in this paragraph, the second temperature selected from the range of about 55-69°C can be about 55°C, 55.5°C, 56°C, 56.5°C, 57°C, 57.5°C, 58°C, or 58.5°C. Temperatures ranged from 59°C to 69°C. Specifically, in any of the embodiments described in this paragraph, the Bst DNA polymerase may be wild-type Bst DNA polymerase, or a Bst DNA polymerase derived from a large fragment of Bst DNA polymerase, Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, or a Bst 3.0 DNA polymerase that has been mutated or truncated.

[0166] In some embodiments, the polymerase is a Bst DNA polymerase, and the first temperature is selected from the range of about 68-78°C, and the second temperature is selected from the range of about 55-69°C. Specifically, in a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 55°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 55.5°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 56°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 56.5°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 57°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 57.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 58°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 58.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 59°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 59.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 60°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 60.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 61°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 61.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 62°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 62.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 63°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 63.5°C.In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 64°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 64.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 65°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 65.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 66°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 66.5°C. In a specific embodiment where the polymerase is Bst DNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 67°C. In a specific embodiment where the polymerase is BstDNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 67.5°C. In a specific embodiment where the polymerase is BstDNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 68°C. In a specific embodiment where the polymerase is BstDNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 68.5°C. In a specific embodiment where the polymerase is BstDNA polymerase, the first temperature is selected from the range of about 68-78°C, and the second temperature is about 69°C. Specifically, in any embodiment described in this paragraph, the first temperature selected from the range of about 68-78°C can be about 68°C, 68.5°C, 69°C, 69.5°C, 70°C, 70.5°C, 71°C, or 71.5°C. 72℃, 72.5℃, 73℃, 73.5℃, 74℃, 74.5℃, 75℃, 75.5℃, 76℃, 76.5℃, 77℃, 77.5℃, or 78℃. Specifically, in any of the embodiments described in this paragraph, the Bst DNA polymerase may be a wild-type Bst DNA polymerase, or a Bst DNA polymerase derived from Bst DNA polymerase, large fragment Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, or a Bst 3.0 DNA mutant or truncated Bst DNA polymerase.

[0167] In some embodiments, the polymerase is a Bst DNA polymerase, and the first temperature is selected from the range of about 72-76°C, and the second temperature is selected from the range of about 61-65°C. Specifically, in a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 72°C, and the second temperature is about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 73°C, and the second temperature is about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 74°C, and the second temperature is about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In a particular embodiment where the polymerase is a Bst DNA polymerase, the first temperature is about 75°C, and the second temperature is about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In specific embodiments where the polymerase is Bst DNA polymerase, the first temperature is about 76°C, and the second temperature is about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In specific embodiments where the polymerase is Bst DNA polymerase, the first temperature is about 76°C, and the second temperature is about 62°C. In specific embodiments where the polymerase is Bst DNA polymerase, the first temperature is about 76°C, and the second temperature is about 61°C. Specifically, in any of the embodiments described in this paragraph, the Bst DNA polymerase may be a wild-type Bst DNA polymerase, or a mutated or truncated Bst DNA polymerase, such as Bst DNA polymerase large fragment, Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, and Bst 3.0 DNA polymerase.

[0168] In some embodiments, the polymerase is Taq DNA polymerase or a truncated or mutant form thereof, and the first temperature is selected from the range of about 70-88°C, and the second temperature is selected from the range of about 58-70°C. Specifically, in a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 70°C, and the second temperature is selected from the range of about 58-70°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 70.5°C, and the second temperature is selected from the range of about 58-70°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 71°C, and the second temperature is selected from the range of about 58-70°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 71.5°C, and the second temperature is selected from the range of about 58-70°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 72°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 72.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 73°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 73.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 74°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 74.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 75°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 75.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 76°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 76.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 77°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 77.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 78°C, and the second temperature is selected from the range of about 58-70°C.In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 78.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 79°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 79.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 80°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 80.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 81°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 81.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 82°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 82.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 83°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 83.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 84°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 84°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 84.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 85°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 85.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 86°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 86.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 87°C, and the second temperature is selected from the range of about 58-70°C.In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 87.5°C, and the second temperature is selected from the range of about 58-70°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is about 88°C, and the second temperature is selected from the range of about 58-70°C. Specifically, in any embodiment described in this paragraph, the first temperature selected from the range of about 58-70°C can be about 58°C, 58.5°C, 59°C, 59.5°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C, 68.5°C, 69°C, 69.5°C, or 70°C. Specifically, in any of the embodiments described in this paragraph, the Taq DNA polymerase may be a wild-type Taq DNA polymerase, or a mutated or truncated Taq DNA polymerase selected from Hot Start Taq DNA polymerase, EpiMark HotStart Taq DNA polymerase, OneTaq DNA polymerase, OneTaq hot-start DNA polymerase, LongAmp Taq DNA polymerase, or LongTaq DNA polymerase.

[0169] In some embodiments, the polymerase is Taq DNA polymerase or a truncated or mutant form thereof, and the first temperature is selected from the range of about 70-88°C, and the second temperature is selected from the range of about 70-88°C. Specifically, in a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 58°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 58.5°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 59°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 59.5°C. In a particular embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 60°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 60.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 61°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 61.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 62°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 62.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 63°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 63.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 64°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 64.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 65°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 65.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 66°C.In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 66.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 67°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 67.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 68°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 68.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 69°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 69.5°C. In a specific embodiment where the polymerase is Taq DNA polymerase, the first temperature is selected from the range of about 70-88°C, and the second temperature is about 70°C. Specifically, in any embodiment described in this paragraph, the Taq DNA polymerase may be a wild-type Taq DNA polymerase, or a mutant or truncated Taq DNA polymerase selected from Hot Start Taq DNA polymerase, EpiMark Hot Start Taq DNA polymerase, OneTaq DNA polymerase, OneTaq hot-start DNA polymerase, LongAmp Taq DNA polymerase, or LongTaq DNA polymerase.

[0170] In some embodiments, the polymerase is DNA polymerase I or a truncated or mutant thereof, and the first temperature is selected from the range of about 50-60°C, and the second temperature is selected from the range of about 30-40°C. Specifically, in a particular embodiment where the polymerase is DNA polymerase I, the first temperature is about 50°C, and the second temperature is selected from the range of about 30-40°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is about 50.5°C, and the second temperature is selected from the range of about 30-40°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is about 51°C, and the second temperature is selected from the range of about 30-40°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is about 51.5°C, and the second temperature is selected from the range of about 30-40°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is about 52°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 52.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 53°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 53.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 54°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 54.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 55°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 55.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 56°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 56.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 57°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 57.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 58°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 58.5°C, and the second temperature is selected from the range of about 30-40°C.In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 59°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 59.5°C, and the second temperature is selected from the range of about 30-40°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is about 60°C, and the second temperature is selected from the range of about 30-40°C. Specifically, in any embodiment described in this paragraph, the second temperature is selected from the range of about 30-40°C, and may be about 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, or 40°C. In particular, in any of the embodiments described in this paragraph, the polymerase may be selected from wild-type DNA polymerase I, DNA polymerase I large fragment (Klenow), or Klenow exo. - .

[0171] In some embodiments, the polymerase is DNA polymerase I or a truncated or mutant form thereof, and the first temperature is selected from the range of about 50-60°C, and the second temperature is selected from the range of about 30-40°C. Specifically, in a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 30°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 30.5°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 31°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 31.5°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 32°C. In a particular embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 32.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 33°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 33.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 34°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 34.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 35°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 35.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 36°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 36.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 37°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 37.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 38°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 38.5°C.In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 39°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 39.5°C. In a specific embodiment where the polymerase is DNA polymerase I, the first temperature is selected from the range of about 50-60°C, and the second temperature is about 40°C. Specifically, in any embodiment described in this paragraph, the second temperature is selected from the range of about 50-60°C, and may be about 50°C, 50.5°C, 51°C, 51.5°C, 52°C, 52.5°C, 53°C, 53.5°C, 54°C, 54.5°C, 55°C, 55.5°C, 56°C, 56.5°C, 57°C, 57.5°C, 58°C, 58.5°C, 59°C, 59.5°C, or 60°C. In particular, in any of the embodiments described in this paragraph, the polymerase may be selected from wild-type DNA polymerase I, DNA polymerase I, large fragment (Klenow), or Klenowexo-.

[0172] In some embodiments, polymerase is DNA polymerase or a truncated or mutant thereof, with the first temperature selected from the range of approximately 70-80°C and the second temperature selected from the range of approximately 55-70°C. Specifically, when the polymerase is... In a specific embodiment of the DNA polymerase, the first temperature is about 70°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 70.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 71°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 71.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 72°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 72.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 73°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 73.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 74°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 74.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 75°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 75.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 76°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 76.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 77°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 77.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 78°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 78.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 79°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 79.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 80°C, and the second temperature is selected from the range of about 55-70°C. Specifically, in any embodiment described in this paragraph, the second temperature selected from the range of about 55-70°C can be about 55°C, 55.5°C, 56°C, 56.5°C, 57°C, 57.5°C, 58°C, 58.5°C, 59°C, 59.5°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C, 68.5°C, 69°C, 69.5°C, or 70°C. In particular, in any of the embodiments described in this paragraph, the polymerase may be Vent DNA polymerase, Vent(exo-) DNA polymerase, Deep Vent DNA polymerase, or DeepVent(exo-) DNA polymerase.

[0173] In some embodiments, the polymerase is a DNA polymerase or a truncated or mutant form thereof, and the first temperature is selected from the range of about 70-80°C, and the second temperature is selected from the range of about 55-70°C. Specifically, when the polymerase is In a specific embodiment of the DNA polymerase, the first temperature is about 70°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 70.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 71°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 71.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 72°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 72.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 73°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 73.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 74°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 74.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 75°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 75.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 76°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 76.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 77°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 77.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 78°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 78.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 79°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 79.5°C, and the second temperature is selected from the range of about 55-70°C. In the polymerase... In a specific embodiment of the DNA polymerase, the first temperature is about 80°C, and the second temperature is selected from the range of about 55-70°C. Specifically, in any embodiment described in this paragraph, the second temperature selected from the range of about 55-70°C can be about 70°C, 70.5°C, 71°C, 71.5°C, 72°C, 72.5°C, 73°C, 73.5°C, 74°C, 74.5°C, 75°C, 75.5°C, 76°C, 76.5°C, 77°C, 77.5°C, 78°C, 78.5°C, 79°C, 79.5°C, or 80°C. Specifically, in any embodiment described in this paragraph, the polymerase can be Vent DNA polymerase, Vent(exo-) DNA polymerase, Deep Vent DNA polymerase, or Deep Vent(exo-) DNA polymerase.

[0174] In some embodiments, the polymerase is a Phi29 DNA polymerase, and the first temperature is selected from the range of about 40-55°C, and the second temperature is selected from the range of about 20-37°C. Specifically, in a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is about 40°C, and the second temperature is selected from the range of about 20-37°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is about 40.5°C, and the second temperature is selected from the range of about 20-37°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is about 41°C, and the second temperature is selected from the range of about 20-37°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is about 41.5°C, and the second temperature is selected from the range of about 20-37°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is about 42°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 42.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 43°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 43.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 44°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 44.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 45°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is about 45.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 46°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 46.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 47°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 47.5°C, and the second temperature is selected from the range of about 20-37°C.In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 48°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 48.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 49°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 49.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 50°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 50.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 51°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 51.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 52°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 52.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 53°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 53.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 54°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is about 54.5°C, and the second temperature is selected from the range of about 20-37°C. In a specific embodiment where the polymerase is phi29 DNA polymerase, the first temperature is about 55°C, and the second temperature is selected from the range of about 20-37°C.

[0175] In some embodiments, the polymerase is a Phi29 DNA polymerase, and the first temperature is selected from the range of about 40-55°C, and the second temperature is selected from the range of about 20-37°C. Specifically, in a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 20°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 20.5°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 21°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 21.5°C. In a particular embodiment where the polymerase is a Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 22°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 22.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 23°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 23.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 24°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 24.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 25°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 25.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 26°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 26.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 27°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 27.5°C.In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 28°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 28.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 29°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 29.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 30°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 30.5°C. In a specific embodiment where the polymerase is PHI29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 31°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 31.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 32°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 32.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 33°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 33.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 34°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 34.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 35°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 35.5°C. In a specific embodiment where the polymerase is Phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 36°C.In a specific embodiment where the polymerase is phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 36.5°C. In a specific embodiment where the polymerase is phi29 DNA polymerase, the first temperature is selected from the range of about 40-55°C, and the second temperature is about 37°C. Specifically, in any embodiment described in this paragraph, the first temperature selected from the range of approximately 40-55°C can be approximately 40°C, 40.5°C, 41°C, 41.5°C, 42°C, 42.5°C, 43°C, 43.5°C, 44°C, 44.5°C, 45°C, 45.5°C, 46°C, 46.5°C, 47°C, 47.5°C, 48°C, 48.5°C, 49°C, 49.5°C, 50°C, 50.5°C, 51°C, 51.5°C, 52°C, 52.5°C, 53°C, 53.5°C, 54°C, 54.5°C, or 55°C.

[0176] In some embodiments, the designed primer pair amplifies a region of the target nucleic acid molecule less than 100 bp in length. In some embodiments, the amplification product generated by this method is less than 90 bp in length. In some embodiments, the amplification product generated by this method is less than 80 bp in length. In some embodiments, the amplification product generated by this method is less than 70 bp in length. In some embodiments, the amplification product generated by this method is less than 60 bp in length. In some embodiments, the amplification product generated by this method is less than 50 bp in length. In some embodiments, the amplification product generated by this method is approximately 20-50 bp in length. In some embodiments, the amplification product generated by this method is approximately 30-50 bp in length. In some embodiments, the amplification product generated by this method is approximately 35-50 bp in length.

[0177] In some embodiments, to reduce the time required for primer extension, primer pairs are designed to amplify short fragments with an amplification length of about 20 bp to about 50 bp. The amplification fragment contains at least a central portion corresponding to a unique sequence in the target nucleic acid molecule, flanked by primer sequences that may be the same as or different from the sequence in the target molecule. For example, in a particular embodiment, the amplification fragment is about 20 bp long. In a particular embodiment, the amplification fragment is about 21 bp long. In a particular embodiment, the amplification fragment is about 22 bp long. In a particular embodiment, the amplification fragment is about 23 bp long. In a particular embodiment, the amplification fragment is about 24 bp long. In a particular embodiment, the amplification fragment is about 25 bp long. In a particular embodiment, the amplification fragment is about 26 bp long. In a particular embodiment, the amplification fragment is about 27 bp long. In a particular embodiment, the amplification fragment is about 28 bp long. In a particular embodiment, the amplification fragment is about 29 bp long. In a particular embodiment, the amplification fragment is about 30 bp long. In a particular embodiment, the amplification fragment is about 31 bp long. In a particular embodiment, the amplification fragment is about 32 bp long. In a specific embodiment, the length of the amplified fragment is approximately 33 bp. In a specific embodiment, the length of the amplified fragment is approximately 34 bp. In a specific embodiment, the length of the amplified fragment is approximately 35 bp. In a specific embodiment, the length of the amplified fragment is approximately 36 bp. In a specific embodiment, the length of the amplified fragment is approximately 37 bp. In a specific embodiment, the length of the amplified fragment is approximately 38 bp. In a specific embodiment, the length of the amplified fragment is approximately 39 bp. In a specific embodiment, the length of the amplified fragment is approximately 40 bp. In a specific embodiment, the length of the amplified fragment is approximately 41 bp. In a specific embodiment, the length of the amplified fragment is approximately 42 bp. In a specific embodiment, the length of the amplified fragment is approximately 43 bp. In a specific embodiment, the length of the amplified fragment is approximately 44 bp. In a specific embodiment, the length of the amplified fragment is approximately 45 bp. In a specific embodiment, the length of the amplified fragment is approximately 46 bp. In a specific embodiment, the length of the amplified fragment is approximately 47 bp. In a specific embodiment, the length of the amplified fragment is approximately 48 bp. In a specific embodiment, the length of the amplified fragment is approximately 49 bp. In a specific embodiment, the length of the amplified fragment is approximately 50 bp.

[0178] In some embodiments, the amplification product has a melting temperature (Tm or Tm value) equal to or lower than about 90°C. In some embodiments, the Tm value of the amplification product is equal to or lower than about 89°C. In some embodiments, the Tm value of the amplification product is equal to or lower than about 88°C. In some embodiments, the Tm value of the amplification product is equal to or lower than about 87°C. In some embodiments, the Tm value of the amplification product is equal to or lower than about 86°C. In some embodiments, the Tm value of the amplification product is equal to or lower than about 85°C. In some embodiments, the Tm value of the amplification product is determined using a computer algorithm based on the amplification product sequence. In some embodiments, the computer-based algorithm is based on the sequence of the amplification product and one or more other conditions of the amplification mixture, such as, but not limited to, Na. + The concentration of Mg 2+ The concentration of nucleic acid molecules in the amplification mixture. In some embodiments, the computer algorithm is selected from NUPACK network tools (www.nupack.org), DNAMelt network (http: / / unafold.rna.albany.edu / ?q=DINAMelt), NOVOPRO (www.novopro.cn / tools / rev_comp.html), the BLAST algorithm on the NCBI website (www.ncbi.nlm.nih.gov / tools / primer-blast), Primer Premier (Premier Biosoft Inc., Canada), AlignMiner (http: / / www.scbi.uma.es / alignminer / ), Oligo (DBA Oligo, Inc., CO, US), PerlPrimer (http: / / perlprimer.sourceforge.net / ), Primer3Web (http: / / bioinfo.ut.ee / primer3 / ), and DNAstar (DNASTAR Inc., WI, US).

[0179] In some embodiments, the relatively short amplification fragments allow for amplification reactions to be carried out by rapidly changing the reaction temperature between a first temperature and a second temperature, thereby generating detectable amounts of amplification fragments in less than 15 minutes.

[0180] Specifically, in some embodiments, the method includes subjecting the amplification mixture to rapid thermal cycling between a first temperature and a second temperature, wherein each thermal cycle lasts for less than about 20 s, or less than about 15 s, or less than about 10 s, or less than about 8 s, or less than about 6 s, or less than about 5 s, or less than about 4 s, or less than about 3 s, or less than about 2 s, or less than about 1 s, or less than about 0.5 s, or less than about 0.1 s.

[0181] In some embodiments, during each thermal cycle, the amplification mixture is incubated at a first temperature for no more than 5 seconds and at a second temperature for no more than 5 seconds. In some embodiments, during each thermal cycle, the amplification mixture is incubated at a first temperature for no more than 2 seconds and at a second temperature for no more than 2 seconds. In some embodiments, during each thermal cycle, the amplification mixture is incubated at a first temperature for less than about 1 second and at a second temperature for less than about 1 second. In some embodiments, during each thermal cycle, the amplification mixture is incubated at a first temperature for about 0.5 seconds and at a second temperature for about 0.5 seconds. In some embodiments, during each thermal cycle, the amplification mixture is incubated at a first temperature for about 0.1 seconds and at a second temperature for about 0.1 seconds.

[0182] In some embodiments, the time to complete each thermal cycle is longer than the sum of the incubation time at the first temperature and the incubation time at the second temperature, because time is required for the reaction temperature to change between the two temperatures; this time interval is referred to herein as the "ramp time" (i.e., the temperature change time). According to the invention, the total heating time in the thermal cycle also includes the time required to lower the reaction temperature from the first temperature to the second temperature and the time required to increase the reaction temperature from the second temperature to the first temperature. In some embodiments, the total temperature change time in the thermal cycle is less than about 10 s. In some embodiments, the total temperature change time in the thermal cycle is less than about 5 s. In some embodiments, the total temperature change time in the thermal cycle is less than about 2 s. In some embodiments, the total temperature change time in the thermal cycle is less than about 1 s. In some embodiments, the total temperature change time in the thermal cycle is less than about 0.5 s. In exemplary embodiments, as... Example 9 As shown, running this method on a microfluidic platform with a temperature variation rate of 8 °C / s produced detectable specific amplification in less than 8 seconds (40 thermal cycles). Other exemplary methods and instruments that can be used in conjunction with this method and system are provided below.

[0183] Early work in the early 1990s established the feasibility of rapid cycling using capillary tubes and hot air for temperature control. Over the past two decades, following the paradigm of PCR methods, extensive work has been done in this field to improve PCR instrumentation, particularly the ability of thermal cyclers to rapidly and accurately control and monitor reaction temperatures. Researchers have explored various methods and techniques to avoid or reduce delays in temperature changes caused by the heat transfer efficiency of conical tube walls, low specific surface areas, or heating large-volume samples. Researchers have further reduced temperature change times by improving thermally conductive materials and designing novel reaction chambers and heating elements.

[0184] In some embodiments, the number of thermal cycles of this method is approximately 20 to 50 cycles. In some embodiments, the number of thermal cycles of this method is at least 20 cycles. In some embodiments, the number of thermal cycles of this method is at least 25 cycles. In some embodiments, the number of thermal cycles of this method is at least 30 cycles. In some embodiments, the number of thermal cycles of this method is at least 35 cycles. In some embodiments, the number of thermal cycles of this method is at least 40 cycles. In some embodiments, the number of thermal cycles of this method is at least 45 cycles. In some embodiments, the number of thermal cycles of this method is at least 50 cycles.

[0185] In some embodiments, the total response time of the method is approximately 2-20 minutes. In some embodiments, the total response time of the method is less than 20 minutes. In some embodiments, the total response time of the method is less than 15 minutes. In some embodiments, the total response time of the method is less than 10 minutes. In some embodiments, the total response time of the method is less than 7 minutes. In some embodiments, the total response time of the method is less than 5 minutes. In some embodiments, the total response time of the method is less than 2 minutes. (Does "less than 2 minutes" contradict the "2-20 minutes" mentioned at the beginning of this paragraph?)

[0186] In some embodiments, the volume of the amplification mixture ranges from 1 to 30 μL. In some embodiments, the amplification mixture is 1 μL. In some embodiments, the amplification mixture is 2 μL. In some embodiments, the amplification mixture is 3 μL. In some embodiments, the amplification mixture is 4 μL. In some embodiments, the amplification mixture is 5 μL. In some embodiments, the amplification mixture is 6 μL. In some embodiments, the amplification mixture is 7 μL. In some embodiments, the amplification mixture is 8 μL. In some embodiments, the amplification mixture is 9 μL. In some embodiments, the amplification mixture is 10 μL. In some embodiments, the amplification mixture is 15 μL. In some embodiments, the amplification mixture is 20 μL. In some embodiments, the amplification mixture is 25 μL. In some embodiments, the amplification mixture is 30 μL. In some embodiments, the method employs a microfluidic device. In some embodiments, the method employs amplification of the mixture droplets.

[0187] The various samples used in this disclosure include, but are not limited to, biological samples isolated from subjects (e.g., blood samples, saliva samples, nasal and oral swabs), samples containing nucleic acid molecules isolated from biological samples, or samples containing synthetic nucleic acid molecules. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. This invention predicts and demonstrates that the method and kit can be used to amplify and detect trace amounts of target nucleic acid molecules present in samples. In a specific embodiment, the amplification mixture contains less than 1.0 × 10⁻⁶ molecules. -12 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of less than 1.0 × 10⁻⁶. -13 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of less than 1.0 × 10⁻⁶. -14 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of no more than 1.0 × 10⁻⁶. -15 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of no more than 1.0 × 10⁻⁶. -16 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of no more than 1.0 × 10⁻⁶. -17 The target nucleic acid of M. In a specific embodiment, the amplification mixture contains a concentration of no more than 1.0 × 10⁻⁶. -18 M's target nucleic acid.

[0188] In a specific embodiment, the amplification mixture contains less than 1.0 × 10⁻⁶. 7 The target nucleic acid molecule was copied. In a specific embodiment, the amplification mixture contained less than 1.0 × 10⁻⁶. 6 The target nucleic acid molecule was copied. In a specific embodiment, the amplification mixture contained less than 1.0 × 10⁻⁶. 5The target nucleic acid molecule was copied. In a specific embodiment, the amplification mixture contained less than 1.0 × 10⁻⁶. 4 The target nucleic acid molecule was copied. In a specific embodiment, the amplification mixture contained less than 1.0 × 10⁻⁶. 3 The target nucleic acid molecule is a copy. In a specific embodiment, the amplification mixture contains fewer than 100 copies of the target nucleic acid molecule. In a specific embodiment, the amplification mixture contains fewer than 10 copies of the target nucleic acid molecule.

[0189] As will be appreciated by those skilled in the art, the methods and systems of the present invention can detect trace amounts of target nucleic acids present in a sample. To prevent potential contamination of the reaction by nucleic acid molecules floating in the ambient air, in some embodiments, the method further includes steps to prevent or reduce the impact of potential contamination.

[0190] Uracil-DNA glycosyltransferase (UDG) is an enzyme that catalyzes the hydrolysis of the N-glycosyl bond between uracil and sugar residues, releasing free uracil and leaving a pyrimidinidone site in uracil-containing single-stranded or double-stranded DNA. UDG is active against DNA containing single-stranded and double-stranded uracil (dU), but dUTPs are not substrates for UDG. UDG can be used to specifically degrade nucleic acids produced by previous amplification reactions (a common source of residual contaminants). In some embodiments, UDG can degrade non-specific products from previous amplification products or incorrect primer extensions, while retaining the native nucleic acid template originally intended for amplification. Therefore, in some embodiments, the amplification mixture contains dUTPs for performing the amplification reaction. In certain embodiments, the amplification mixture contains uracil-DNA glycosyltransferase (UDG) for the reaction. In certain embodiments, the amplification mixture contains both dUTPs and UDG for the reaction. In some embodiments where dUTPs are used for amplification, the amplification mixture does not contain dUTPs.

[0191] The primers here can be used in conjunction with this method, for example... Section 4.3 The primers described and according to the example Example 7 The exemplary process described describes primers designed for specific purposes. Particularly in some embodiments, the amplification reaction comprises a pair of primers for identifying an approximately 20–50 bp amplification region of the target nucleic acid molecule. Particularly in some embodiments, the concentration of at least one primer present in the amplification mixture is not less than 1.0 × 10⁻⁶. -6 M. In some embodiments, the concentration of at least one primer present in the amplification mixture is not less than 1.5 × 10⁻⁶. -6 M. In some embodiments, the concentration of at least one primer present in the amplification mixture is not less than 2.0 × 10⁻⁶. -6M. In some embodiments, the concentration of at least one primer present in the amplification mixture is not less than 2.5 × 10⁻⁶. -6 M. In some embodiments, the concentration of at least one primer present in the amplification mixture is not less than 3.0 × 10⁻⁶. -6 M. In some embodiments, the concentration of all primers present in the amplification mixture is not less than 1.0 × 10⁻⁶. -6 M. In some embodiments, the concentration of all primers present in the amplification mixture is not less than 1.5 × 10⁻⁶. -6 M. In some embodiments, the concentration of all primers present in the amplification mixture is not less than 2.0 × 10⁻⁶. -6 M. In some embodiments, the concentration of all primers present in the amplification mixture is not less than 2.5 × 10⁻⁶. -6 M. In some embodiments, the concentration of all primers present in the amplification mixture is not less than 3.0 × 10⁻⁶. -6 M.

[0192] Specifically, in some embodiments, the melting temperature (Tm or Tm value) of one primer is within ±6°C of the second temperature used in the method. The Tm value of one primer is within ±5°C of the second temperature used in the method. In some embodiments, the Tm value of one primer is within ±4°C of the second temperature used in the method. In some embodiments, the Tm value of one primer is within ±3°C of the second temperature used in the method. In some embodiments, the Tm value of one primer is within ±2°C of the second temperature used in the method. In some embodiments, the Tm value of one primer is within ±1°C of the second temperature used in the method. In some embodiments, the Tm value of one primer is within ±0.5°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±6°C of the second temperature used in the method. The Tm values ​​of all primers are within ±5°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±4°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±4°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±3°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±1°C of the second temperature used in the method. In some embodiments, the Tm values ​​of all primers are within ±0.5°C of the second temperature used in the method.

[0193] In some embodiments, the first temperature used in the method is within ±6°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±5°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±4°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±3°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±2°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±1°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is within ±0.5°C of the Tm value of the amplification product. In some embodiments, the first temperature used in the method is approximately the same as the Tm value of the amplification product. In the specific embodiments described in this paragraph, the second temperature used in the method is within ±6°C of the Tm value of at least one primer. In the specific embodiments described in this paragraph, the second temperature used in the method is at least within ±6°C, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C, or ±0.5°C of the Tm value of one primer. In the specific embodiments described in this paragraph, the second temperature used in the method is approximately the same as the Tm value of at least one primer used in the method. In the specific embodiments described in this paragraph, the Tm values ​​of a pair of primers are approximately the same. In the specific embodiments described in this paragraph, the Tm values ​​of a pair of primers differ from each other by less than about 3°C, about 2°C, about 1°C, or about 0.5°C.

[0194] In some embodiments, each of the primer pair used in this method has a melting temperature. The average of the two Tm values ​​of the primer pair is called the average melting temperature of the primer pair. Specifically, in some embodiments, the second temperature used in this method is within ±6°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±5°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±4°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±3°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±2°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±1°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is within ±0.5°C of the average melting temperature of the primer pair used in this method. In some embodiments, the second temperature used in this method is substantially the same as the average melting temperature of the primer pair used in this method. In the specific embodiments described in this paragraph, the Tm values ​​of a pair of primers are approximately the same. In the specific embodiments described in this paragraph, the Tm values ​​of a pair of primers differ from each other by less than about 3°C, about 2°C, about 1°C, or about 0.5°C. In the specific embodiments described in this paragraph, the first temperature used in the method is within ±6°C, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C, or ±0.5°C of the Tm value of the amplification product. In the specific embodiments described in this paragraph, the first temperature used in the method is approximately the same as the Tm value of the amplification product.

[0195] Methods for determining the Tm value of nucleic acids (e.g., oligonucleotide primers or amplification products) are known in the art. For example, various computer algorithms are known in the art that can determine the Tm value based on the nucleic acid sequence and / or environmental conditions (e.g., salt concentration). Exemplary computer algorithms or software that can be used to determine the Tm value of nucleic acids include, but are not limited to, the NUPACK network tool (www.nupack.org), the DNAMelt network (http: / / unafold.rna.albany.edu / ?q=DINAMelt), NOVOPRO (www.novopro.cn / tools / rev_comp.html), the BLAST algorithm on the NCBI website (www.ncbi.nlm.nih.gov / tools / primer-blast), Primer Premier (Premier Biosoft Inc., Canada), AlignMiner (http: / / www.scbi.uma.es / alignminer / ), Oligo (DBA Oligo, Inc., CO, US), PerlPrimer (http: / / perlprimer.sourceforge.net / ), Primer3Web (http: / / bioinfo.ut.ee / primer3 / ), and DNAstar (DNASTAR Inc., WI, US). In some implementations, the computer algorithms or software used to determine the Tm value of nucleic acids are selected from NUPACK Network Tools (www.nupack.org), DNAMelt Network (http: / / unafold.rna.albany.edu / ?q=DINAMelt), NOVOPRO (www.novopro.cn / tools / rev_comp.html), and the BLAST algorithm on the NCBI website (www.ncbi.nlm.nih.gov / tools / primer-blast).

[0196] In some embodiments, the method further includes determining the Tm value of the amplification product to be generated by the method. In some embodiments, the method further includes determining the Tm value of at least one primer used in the method. In some embodiments, the method further includes determining the Tm values ​​of two primers to be used in the method. In some embodiments, the method further includes determining the Tm values ​​of two primers to be used in the method, and further includes determining the average melting temperature of a pair of primers to be used in the method. In some embodiments, the Tm value of the primer or amplification product is determined using a computer algorithm based on the sequence of the primer or amplification product. In some embodiments, the Tm value is determined based on the sequence of the primer or amplification product and one or more other conditions of the amplification mixture, such as, but not limited to, Na+. + The concentration of Mg 2+ The concentration of primers or nucleic acid molecules in the amplification mixture is used to determine the Tm value of primers or amplification products using computer algorithms.

[0197] The polymerase mentioned here can be used in conjunction with this method, for example... Section 4.4 The polymerase described herein. Specifically, in some embodiments, the polymerase is the thermostable polymerase described herein. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.1 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.2 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.3 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.4 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 0.5 U / μL. In some embodiments, the amplification mixture contains a polymerase at a concentration of not less than 1 U / μL.

[0198] In some embodiments, the optimal temperature of the polymerase is between the first temperature and the second temperature used in this method. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 6°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 5°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 4°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 3°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 2°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 1°C. In some embodiments, the optimal temperature of the polymerase is the second temperature used in the method ± 0.5°C.

[0199] In some embodiments, the optimal temperature for the polymerase is ±5°C of the first temperature used in the method. In some embodiments, the optimal temperature for the polymerase is ±4°C of the first temperature used in the method. In some embodiments, the optimal temperature for the polymerase is ±3°C of the first temperature used in the method. In some embodiments, the optimal temperature for the polymerase is ±2°C of the first temperature used in the method. In some embodiments, the optimal temperature for the polymerase is ±1°C of the first temperature used in the method. In some embodiments, the optimal temperature for the polymerase is ±0.5°C of the first temperature used in the method.

[0200] In one specific embodiment, the present invention discloses a method for amplifying target nucleic acid molecules in a sample: Bst DNA polymerase and a pair of oligonucleotide primers are contacted with the sample to form an amplification mixture. Multiple thermal cycles are performed at a first temperature selected from approximately 76°C, approximately 75°C, approximately 74°C, approximately 73°C, and approximately 72°C, and a second temperature selected from approximately 61°C, approximately 62°C, approximately 63°C, approximately 64°C, and approximately 65°C. Each thermal cycle includes incubating the amplification mixture at the first temperature for no more than 1 second and incubating the amplification mixture at the second temperature for no more than 1 second, with a total incubation time not exceeding 2 seconds, thereby generating an amplified fragment of approximately 20-50 base pairs (bp) in length within 10 minutes. Specifically, in this embodiment, the concentration of the target nucleic acid in the sample is less than 1.0 × 10⁻⁶. -14 M. More specifically, in this embodiment, the concentration of the target nucleic acid in the sample is less than 1.0 × 10⁻⁶. -15 M, less than 1.0 × 10 -16 M, less than 1.0 × 10 -17 M or less than 1.0 × 10 -18 M. Specifically, in this embodiment, the concentration of the target nucleic acid in the sample is less than 1.0 × 10⁻⁶. 5 Copy. More specifically, in this embodiment, the concentration of the target nucleic acid in the sample is less than 1.0 × 10⁻⁶. 4 Copy, less than 1.0×10 3 Copy, less than 100 copies or less than 10 copies.

[0201] The amplified fragments produced by this method can be detected using methods known in the art, such as fluorescence detection, colorimetric detection, and electrophoresis. Conventional methods for real-time monitoring of PCR amplification can also be used for real-time monitoring of amplification using this method. Specifically, in some embodiments, the amplification level can be detected during each thermal cycle. In other embodiments, the amplification level is detected every 2, 5, or 10 thermal cycles. The amplification products can be purified from the amplification mixture and subjected to sequence analysis, such as next-generation sequencing, to determine the sequence, origin, and molecular properties of the target nucleic acid.

[0202] Such detection and analysis of amplified products can further serve as the basis for various analyses and diagnoses related to the target nucleic acid and its source (e.g., the biological sample containing the target nucleic acid and the subject providing the biological sample). As a non-limiting example, the methods and kits disclosed herein can be used to detect the presence of pathogens in biological samples. For example, the methods and kits can design primers targeting amplification regions of unique sequences in the genome of a pathogen to detect the presence of unique sequences in a biological sample. For example, these methods can be used to diagnose infectious diseases caused by pathogens, detect adulteration or pathogen contamination in biological samples, and for quality control of food and beverages. As another non-limiting example of the disclosure herein, the methods and kits disclosed herein can be used to detect genetic alterations in subjects. Specifically, this includes, but is not limited to, the detection of single nucleotide polymorphisms in subjects and genetic diseases attributed to point mutations. For example, the methods and kits can design primers targeting amplification regions in genomic sequences known to or prone to such mutations, and detect the presence of mutations through sequencing analysis of the amplified products. Other possible applications of the methods and kits disclosed herein will be readily understood by those skilled in the art after reading this disclosure, and other possible uses and applications are also contemplated and included in this disclosure.

[0203] Therefore, in another aspect, this document provides a method for detecting target nucleic acids in a sample, the method comprising mixing a polymerase and a pair of oligonucleotide primers with the sample to form an amplification mixture, subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify at least a portion of the target nucleic acid by polymerase chain reaction, and detecting the presence of amplification products in the amplification mixture. In some embodiments, the first temperature is selected from the range of about 68°C to 78°C. In some embodiments, the second temperature is selected from the range of about 55°C to 69°C. In some embodiments, the pair of oligonucleotide primers is designed to produce an amplification product of about 20-50 bp in length. In some embodiments, the polymerase is selected from Bst DNA polymerase, DNA polymerase I large fragment (Klenow), and... DNA polymerase, or a mutated or truncated form thereof. In some embodiments, the amplification mixture also contains dNTPs and polyethylene glycol. In some embodiments, the detection of the amplification products is performed by fluorescence detection, colorimetric detection, or other methods known in the art. For example, in some embodiments, the method also provides real-time monitoring of the amplification. Particularly in some embodiments, the amount of amplification produced is measured every 1 thermal cycle. In other embodiments, the amount of amplification is detected every 2, 5, or 10 thermal cycles. The detection and measurement of the quantity of amplification products can be achieved using conventional real-time monitoring PCR amplification methods.

[0204] On the other hand, the present invention provides a method for diagnosing pathogen infection in a subject, the method comprising contacting a polymerase and a pair of oligonucleotide primers with a sample to form an amplification mixture, the oligonucleotide primers being designed to amplify a unique sequence in the pathogen genome; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to generate an amplification product by a polymerase chain reaction; and detecting the presence of the amplification product in the amplification mixture. In some embodiments, the first temperature is selected from the range of about 68°C to 78°C. In some embodiments, the second temperature is selected from the range of about 55°C to 69°C. In some embodiments, the length of the amplification product is about 20-50 bp. In some embodiments, the polymerase is selected from Bst DNA polymerase, DNA polymerase I large fragment (Klenow), and... DNA polymerase, or a mutated or truncated form thereof. In some embodiments, the amplification mixture further comprises dNTPs and polyethylene glycol. In some embodiments, the sample is the genomic nucleic acid of a subject. In some embodiments, the sample is a cell-free nucleic acid molecule of a subject. In some embodiments, the sample is a bodily fluid sample. In some embodiments, the pathogen is a microorganism, such as a virus, bacteria, or fungus. In some embodiments, the pathogen is a parasite, such as a protozoan, worm, or ectoparasite.

[0205] On the other hand, this article provides a method for detecting genetic alterations in a subject, comprising contacting a polymerase and a pair of oligonucleotide primers with a sample to form an amplification mixture, the oligonucleotide primers being engineered to amplify a target sequence having or suspected of having a genetic alteration; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to generate amplification products via a polymerase chain reaction; and sequencing the amplification products to determine the presence of a genetic alteration. In some embodiments, the first temperature is selected from the range of about 68°C to 78°C. In some embodiments, the second temperature is selected from the range of about 55°C to 69°C. In some embodiments, the length of the amplicon is about 20-50 bp. In some embodiments, the polymerase is selected from Bst DNA polymerase, DNA polymerase I large fragment (Klenow), and... DNA polymerase, or a mutated or truncated form thereof. In some embodiments, the amplification mixture further comprises dNTPs and polyethylene glycol. In some embodiments, the genetic alteration is a gene mutation, such as an insertion, deletion, substitution, or copy number variation. In some embodiments, the genetic alteration is a single nucleotide polymorphism. In some embodiments, the method further includes the diagnosis or prognosis of a genetic condition associated with the genetic alteration.

[0206] 4.6 Reagent Kit

[0207] On the other hand, this disclosure also provides a kit for carrying out this method. The kit contains multiple components that are mixed together in an amplification mixture or contained in at least two separate containers. In some embodiments, the kit contains a polymerase and a pair of nucleotide primers. The primers provided herein (e.g., in…) Section 4.3 The primers described in the text and based on Reality Example 7 The exemplary program design primers described herein and the polymerases provided herein (e.g., in...) Section 4.4 The polymerase described in [the kit] can be used in conjunction with this kit.

[0208] In some embodiments, the kit contains dNTPs and a buffer solution suitable for the polymerase. The buffer solution provides ion concentrations, pH, and / or coenzymes that promote polymerase activity. Methods for selecting and preparing buffer solutions suitable for a particular polymerase are known in the art. For example, commercially available polymerases are typically sold with recommended formulations of appropriate buffers. In some embodiments, the kit also contains polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is PEG 200, PEG 400, PEG 2000, or PEG 4000. In some embodiments, the kit also contains glycerol.

[0209] In some embodiments, the kit further comprises a reagent, such as a single-stranded binding protein (SSB), capable of promoting the dissociation of double strands near the primer annealing site in the target nucleic acid. In some embodiments, the SSB is stable and active within the temperature range in which the method is performed. In specific embodiments, the SSB is derived from microorganisms, such as bacteria or bacteriophages. In specific embodiments, this kit contains a selection from T4 phage 32SSB, T7 phage 2.5SSB, phage 29SSB, or Escherichia coli SSB.

[0210] In some embodiments, the kit also includes reagents for detecting and quantifying the amplification products, such as fluorescent dyes or pH indicators. Reagents suitable for this purpose are known in the art; for example, certain fluorescent dyes (e.g., Evagreen) emit a stronger fluorescence signal when bound to double-stranded amplification products, and the intensity of the fluorescence signal emitted by the amplification reaction can reflect the amount of amplification products produced.

[0211] In some embodiments, the kit further includes instructions for using the kit. For example, in some embodiments, the various components of the kit are provided as a mixture, and the kit includes instructions for adding appropriate amounts of sample to form an amplification mixture. Alternatively, in some embodiments, the various components of the kit are provided in at least two separate containers, and the kit includes instructions for mixing the components with appropriate amounts of sample in separate containers to form an amplification mixture.

[0212] In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 0.1 U / μL. In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 0.2 U / μL. In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 0.3 U / μL. In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 0.4 U / μL. In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 0.5 U / μL. In specific embodiments, the specification indicates that the amplification mixture contains polymerase at a concentration of not less than 1 U / μL.

[0213] In a specific embodiment, the specification states that the amplification mixture contains a concentration of not less than 1.0 × 10⁻⁶. -6 At least one primer of M. In a specific embodiment, the specification indicates that the amplification mixture contains a concentration of not less than 1.5 × 10⁻⁶. -6 At least one primer of M. In a specific embodiment, the specification indicates that the amplification mixture contains a concentration of not less than 2.0 × 10⁻⁶. -6 At least one primer of M. In a specific embodiment, the specification indicates that the amplification mixture contains a concentration of not less than 2.5 × 10⁻⁶. -6 At least one primer of M. In a specific embodiment, the specification indicates that the amplification mixture contains a concentration of not less than 3.0 × 10⁻⁶. -6 At least one primer for M.

[0214] In a specific embodiment, the specification states that the amplification mixture contains two primers, each with a concentration of not less than 1.0 × 10⁻⁶. -6 M. In a specific embodiment, the specification indicates that the amplification mixture contains two primers, each with a concentration of not less than 1.5 × 10⁻⁶. -6 M. In a specific embodiment, the specification indicates that the amplification mixture contains two primers, each with a concentration of not less than 2.0 × 10⁻⁶. -6 M. In a specific embodiment, the specification indicates that the amplification mixture contains two primers, each with a concentration of not less than 2.5 × 10⁻⁶. -6 M. In a specific embodiment, the specification states that the amplification mixture contains two primers, each with a concentration of not less than 3.0 × 10⁻⁶. -6 M.

[0215] In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶. -13 The target nucleic acid of M. In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶ nucleic acids. -14The target nucleic acid of M. In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶ nucleic acids. -15 The target nucleic acid of M. In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶ nucleic acids. -16 The target nucleic acid of M. In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶ nucleic acids. -17 The target nucleic acid of M. In a specific embodiment, the specification states that the amplification mixture should contain at least 1.0 × 10⁻⁶ nucleic acids. -18 The target nucleic acid of M. In a specific embodiment, the specification indicates that the amplification mixture contains no more than 10 copies of the target nucleic acid molecule.

[0216] In specific embodiments, the specification indicates that the amplification mixture contains at least 0.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains about 0.5%-10% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 0.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 1% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 1.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 2% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 2.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 3% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 3.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 4% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 4.5% PEG by volume. In specific embodiments, the specification indicates that the amplification mixture contains at least about 5% PEG by volume. In a specific embodiment, the specification indicates that the amplification mixture contains at least about 10% PEG by volume.

[0217] In specific embodiments, the specification indicates that the amplification mixture contains approximately 1-50 μg / mL of SSB. In specific embodiments, the specification indicates that the amplification mixture contains approximately 1 μg / mL of SSB. In specific embodiments, the specification indicates that the amplification mixture contains approximately 5 μg / mL of SSB. In specific embodiments, the specification indicates that the amplification mixture contains approximately 12.5 μg / mL of SSB. In specific embodiments, the specification indicates that the amplification mixture contains approximately 25 μg / mL of SSB. In specific embodiments, the specification indicates that the amplification mixture contains approximately 50 μg / mL of SSB.

[0218] In a specific embodiment, the specification states that the volume of the amplification mixture is approximately 1-30 μL. In a further specific embodiment, the specification indicates that the amplification mixture can be loaded onto a microfluidic device for PCR reaction.

[0219] In some embodiments, the kit further includes instructions for performing PCR on the amplification mixture under a thermal cycling protocol. In a specific embodiment, the thermal cycling protocol includes multiple thermal cycles, each including incubation at a first temperature and incubation at a second temperature. In a specific embodiment, the first temperature range is 68-78°C, and the second temperature range is 55-69°C. In some embodiments, each thermal cycle also includes a temperature change time of less than 10 seconds. In one specific embodiment, the thermal cycling protocol includes incubation at a first temperature in the range of about 72-76°C for about 1 second, incubation at a second temperature in the range of about 61-65°C for about 1 second, and a temperature rise time of less than 2 seconds, with a total reaction time of less than 8 minutes. Example

[0220] The embodiments described below in relation to the present invention may be substituted with other techniques in most cases. The embodiments are intended to illustrate, not limit, the scope of the invention. For example, when preparing the PCR reaction system according to the described scheme, conditions may vary; for example, any solvent, reaction time, reagents, temperature, supplements, reaction conditions, or other reaction parameters may be varied. For example, different methods may be used to detect the PCR amplification process of the target sequence, and the amplification mixture may not contain fluorescent dyes if real-time monitoring of PCR amplification is not required. Moreover, although the nucleic acid targets to be detected in the following embodiments are derived from microorganisms, the application of the current methods and systems is not limited to this, but can be applied to the detection of other types of genetic samples, such as genetic material derived from mammals. Furthermore, although kit components are used in the examples below, these specific designs are not unique or optimal. Reagent types, volumes, concentrations, and packaging are also variable. It should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described, as these may vary depending on the context and the skill of the person skilled in the art.

[0221] General method: Unless otherwise stated, the methods and apparatus used in the following examples are standard methods and apparatus used in similar studies in the relevant art. Unless otherwise stated, the test materials used in the following examples were purchased from biochemical reagent stores or other commercial suppliers. All molecular biology and PCR reactions involving biomolecules such as DNA, RNA, and proteins used standardized plates, vials, and EP tubes. Commercial reagents were used as required.

[0222] In the following examples, genomic DNA or RNA samples were extracted using a DNA / RNA extraction kit purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China, catalog number DP422). The isothermal reaction buffer was prepared using purified water with the following solute concentrations: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100; pH 8.8 at 25°C. Quantitative results were based on at least three replicate experiments.

[0223] 5.2 Example 1: Primer concentration optimization for the rapid chain substitution amplification (SEA) reaction system.

[0224] The following study was conducted to test the effect of primer concentration on the amplification rate of the fast SEA reaction.

[0225] Based on the target nucleic acid sequence of the hypervariable region encoding the 16S rRNA gene of Listeria monocytogenes, a pair of specific primers was designed using NUPACK software (www.nupack.org / ). The target sequence is a 50bp synthetic fragment with the following sequence:

[0226] 5'-GGGTCATTGGAAACTGGAAGACTGGAGTGCAGAAGAGGAGAGTGGAATTC-3'(SEQ ID NO:1),

[0227] The primer sequences are as follows:

[0228] Primer 1:5'-GTCATTGGAAACTGGAAGACTG-3'(M58822.1 b)(SEQ ID NO:2);

[0229] Primer 2:5'-CCACTCTCCTCTTCTGCAC-3'(M58822.1 b) (SEQ ID NO:3).

[0230] Primers and target fragments were chemically synthesized (Sangon Biotech Co., Ltd., Shanghai, China). DNA polymerase, dNTPs solution, other buffer solutions, fluorescent dyes (e.g., Evagreen), and strand displacement amplification (SEA) detection kit were purchased from NED Biotech Co., Ltd. (Qingdao, China).

[0231] The synthesized primers and Listeria monocytogenes genomic material were then mixed with other PCR reactants to form a 10 μL amplification mixture, as shown in Table 1. To improve the amplification rate, four amplification systems with different primer concentrations were prepared for primer concentration optimization. Each mixture contained polymerase at a final concentration of 0.24 U / μL and polymerase at a final concentration of 1.5 × 10⁻⁶. -6 M, 2.0×10 -6 M, 2.5×10 -6 M and 3.0×10 -6 M primers. The negative control group (NTC) refers to an amplification system in which an equal amount of water is used instead of Listeria monocytogenes genomic material, while all other components remain the same.

[0232] Table 1: Composition of the amplification mixture used to optimize primer concentration

[0233]

[0234] To enable rapid SEA response, use CFX Connect. TM The real-time PCR system (Bio-Rad, CA) enables rapid thermal cycling of the amplification mixture between 76°C and 62°C. Each thermal cycle consists of the following steps: first, the amplification mixture is incubated at 76°C for 1 second, then immediately cooled to 62°C for 1 second, and then the temperature is raised back to 76°C. To monitor amplification in real time and reduce fluorescence readout time, the fluorescence signal of the amplification mixture is scanned every two thermal cycles, and a fluorescence curve is plotted as a function of time. Figure 2 ).

[0235] As shown in the figure, for each primer concentration, the rapid SEA reaction achieved target sequence detection within 20 minutes. In particular, increasing the primer concentration to 3.0 × 10⁻⁶ was effective. -6 When M is used, amplification efficiency and rate can be significantly improved, thereby reducing the time required for target nucleic acid detection in samples to less than 15 minutes.

[0236] 5.3 Example 2: Optimization of polymerase concentration for a rapid chain displacement amplification (SEA) reaction system.

[0237] The following study was conducted to test the effect of polymerase concentration on the rate of fast SEA reaction.

[0238] As described in Example 1 above, identical primers (SEQ ID NO: 1 and 2) were designed for the same target sequence in the Listeria monocytogenes genome (SEQ ID NO: 1). The primers and Listeria monocytogenes genome were prepared as described above and mixed with other PCR reactants to form a 10 μL amplification mixture, as shown in Table 2 below. To achieve the optimal polymerase concentration for the amplification rate, four amplification mixtures containing different enzyme concentrations were prepared, each containing a final concentration of 3.0 × 10⁻⁶. -6 Primers for M and polymerase at final concentrations of 0.16 U / μL, 0.20 U / μL, 0.24 U / μL, and 0.28 U / μL (corresponding to 0.20 μL, 0.25 μL, 0.30 μL, and 0.35 μL of 8 U / μL enzyme stock solution, respectively). An amplification mixture with an equal volume of water instead of Listeria monocytogenes genomic material and all other components identical served as a negative control (NTC).

[0239] Table 2: Components of the rapid SEA amplification system used to optimize polymerase concentration

[0240]

[0241]

[0242] To enable rapid SEA response, use CFX Connect. TM The real-time PCR system (Bio-Rad, CA) rapidly thermally cycles the amplification mixture between 76°C and 62°C. Each thermal cycle consists of the following steps: first, the amplification mixture is incubated at 76°C for 1 second, then cooled to 62°C and incubated for 1 second, before being restored to 76°C. To monitor amplification in real time and shorten the fluorescence scanning time, the fluorescence signal of the amplification mixture is scanned every two thermal cycles, and a fluorescence curve is plotted as a function of time. Figure 3 ).

[0243] As shown in the figure, for reactions containing polymerase at concentrations of 0.24 U / μL or 0.28 U / μL, the rapid SEA reaction produced detectable target sequence amplification in less than 20 minutes. Increasing the polymerase concentration from 0.24 U / μL to 0.28 U / μL further significantly improved amplification efficiency and rate, reducing the time required for target nucleic acid detection in samples from within 15 minutes to less than 10 minutes.

[0244] 5.4 Example 3: Thermal cycling optimization of a fast chain substitution amplification (SEA) reaction system.

[0245] The following study was conducted to test the effect of denaturation temperature on the amplification efficiency and amplification rate of the fast SEA reaction.

[0246] The amplification system was prepared as described in Example 1 above, wherein the primer concentration was maintained at 3.0 × 10⁻⁶. -6 M, the polymerase concentration was maintained at 0.24 U / μL. The amplification system was then subjected to different thermal cycles for PCR reactions, and the effects of different temperatures on amplification efficiency and rate were evaluated.

[0247] In each thermal cycle, the amplification system was incubated at a higher denaturation temperature for 1 second, followed immediately by an incubation at a lower extension temperature for 1 second. The lower extension temperature can be selected based on the chosen DNA polymerase. In these studies, the extension temperature was set to 62°C, at which Bst DNA polymerase activity was optimal. Without being bound by theory, it can be expected that slight temperature differences may significantly affect the rate and duration of denaturation bubble opening in double-stranded nucleic acid samples, thereby affecting the efficiency and rate of amplification. In these studies, five denaturation temperatures—74°C, 75°C, 76°C, 77°C, and 78°C—were tested and compared. The negative control group (NTC) was identical in composition and content except that water was used instead of Listeria monocytogenes genomic material.

[0248] For example, each thermal cycle between 76°C and 62°C consists of incubating the amplification system at 76°C for 1 second, immediately lowering the temperature to 62°C for 1 second, and then immediately raising the temperature back to 76°C. For each fast SEA reaction, the thermal cycle is repeated at least 35 times. To monitor amplification in real time, the fluorescence signal emitted by the amplification system is scanned every two thermal cycles, and a fluorescence curve is plotted as a function of time. Figure 4 ).

[0249] As shown in the figure, when the denaturation temperature is between 74℃ and 76℃, the rapid SEA reaction can detect the amplification signal of the target sequence within 20 minutes. Among all test temperatures, a denaturation temperature of 76℃ is the optimal temperature, thus minimizing the detection time required for the target nucleic acid in the sample.

[0250] 5.5 Implementation Case 4: Amplification and Detection of DNA Molecules in Samples.

[0251] The following study was conducted to test the ability of a rapid SEA method to detect DNA molecules in biological samples.

[0252] A pair of specific primers was designed based on the target nucleic acid sequence using NUPACK software (www.nupack.org / ). The target sequence is:

[0253] 5'-GGGTCATTGGAAACTGGAAGACTGGAGTGCAGAAGAGGAGAGTGGAATTC-3'(SEQ ID NO:1),

[0254] The primer sequences are as follows:

[0255] Primer 1:5'-GTCATTGGAAACTGGAAGACTG-3'(M58822.1 b)(SEQ ID NO:2);

[0256] Primer 2:5'-CCACTCTCCTCTTCTGCAC-3'(M58822.1 b) (SEQ ID NO:3).

[0257] Primers and target DNA fragments were chemically synthesized (Sangon Biotech Co., Ltd., Shanghai, China) and mixed with other PCR reactants to form a 10 μL amplification system, as shown in Table 3 below. Specifically, two amplification mixtures were prepared, each containing 1.0 × 10⁻⁶ molecules. -12 The target DNA fragment for M was synthesized, or 0.8 ng / μL of Listeria monocytogenes genomic material was used. The primer concentration was 3.0 × 10⁻⁶. -6 M, with a polymerase concentration of 0.24 U / μL, also included an amplification mixture with an equal volume of water instead of Listeria monocytogenes genomic material, and other components identical, serving as a negative control group (NTC).

[0258] Table 3: Composition of DNA Amplification System

[0259] name Final concentration Primer 1 <![CDATA[3.0×10 -6 M]]> Primer 2 <![CDATA[3.0×10 -6 M]]> Synthetic target DNA <![CDATA[1.0×10 -12 M]]> dNTPs 8mM Isothermal reaction buffer 1× Evagreen 20× ET SSB 5μg / mL Bst DNA polymerase 0.24 U / μL Polyethylene glycol 200 100％ pure water Add to 10μL

[0260] To enable rapid SEA response, use CFX Connect. TM The real-time PCR system (Bio-Rad, CA) rapidly thermally cycles the amplification system between 76°C and 62°C. Each thermal cycle consists of the following steps: first, the amplification system is incubated at 76°C for 1 second, then cooled to 62°C and incubated for 1 second, and then restored to 76°C. To monitor amplification in real time and shorten the fluorescence scanning time, the fluorescence signal of the amplification system is scanned every two thermal cycles, and a fluorescence curve as shown in the figure is plotted as a function of time. Figure 5 ).

[0261] As shown in the figure, the rapid SEA method can effectively detect Listeria monocytogenes-related synthetic DNA fragments and genomic nucleic acids within 10 minutes at the provided target concentration, indicating that this method and kit can be used for the immediate diagnosis of pathogen infection.

[0262] 5.6 Example 5: Amplification and detection of RNA molecules in samples.

[0263] The following studies were conducted to test the ability of a rapid SEA method to detect RNA molecules in biological samples.

[0264] Specifically, the same primers (SEQ ID NO: 2 and 3) were designed as described above for target RNA sequences having the following sequences.

[0265] 5'-GGGTCAUUGGAAACUGGAAGACUGGAGUGCAGAAGAGGAGAGUGGAAUUC-3'(SEQ ID NO:7)

[0266] Primers were prepared and RNA target molecules were synthesized as described above. These were then mixed with other PCR reactants to form a 10 μL amplification system, as shown in Table 4 below. The amplification mixture consisted of three parts, each containing 3.0 × 10⁻⁶ molecules. -6 Primer M, polymerase at a concentration of 0.24 U / μL, and polymerase at a concentration of 1.0 × 10⁻⁶. -12 The target RNA molecule of M. An amplification mixture with an equal volume of water instead of Listeria monocytogenes genomic material, and all other components identical, served as a negative control (NTC).

[0267] Table 4: Composition of RNA amplification system

[0268] name Final concentration Primer 1 <![CDATA[3.0×10 -6 M]]> Primer 2 <![CDATA[3.0×10 -6 M]]> Synthesize target RNA <![CDATA[1.0×10 -12 M]]> dNTPs 8mM Isothermal reaction buffer 1× Evagreen 20× ET SSB 5μg / mL Bst DNA polymerase 0.24 U / μL Polyethylene glycol 200 100％ pure water Add to 10μL

[0269] To perform a rapid SEA reaction, the amplification system was rapidly thermally cycled between 76°C and 62°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA). Each thermal cycle consisted of the following steps: first, the amplification system was incubated at 76°C for 1 second, and immediately afterward, it was incubated at 62°C for another 1 second, before the temperature was raised back to 76°C. To monitor the amplification in real time, the fluorescence signal of the amplification system was scanned every two thermal cycles, and a fluorescence curve was plotted as a function of time. Figure 6A ).

[0270] As shown in the figure, using Bst DNA polymerase with reverse transcriptase activity, the rapid SEA method can efficiently detect the target RNA molecules at the stated concentration within approximately 10 minutes. In three replicate control reactions, the amplification products reached the exponential phase at approximately the same time, and no amplification was detected in the negative control group, indicating that the method and reaction system have high reproducibility and stability.

[0271] Finally, to verify that the observed increase in fluorescence signal corresponded to the specific amplification of the target RNA molecule, the amplification products were examined by electrophoresis using a 12.5% ​​polyacrylamide gel after the reaction. The electrophoresis results are as follows: Figure 6BAs shown, the rapid SEA reaction produced the expected amplified fragment (43 bp in length), and the negative reaction (NTC) did not produce the target band. Lane M is a gradient molecular weight DNA ladder, and the corresponding bands for DNA fragments of 20 bp and 40 bp are marked in the figure.

[0272] 5.7 Example 6: Comparison of isothermal SEA reaction under isothermal conditions and fast SEA reaction under rapid thermal cycling conditions

[0273] The following study compares isothermal SEA reactions carried out under isothermal conditions (as described in CN 109136337A) with fast SEA reactions under current rapid thermal cycling conditions.

[0274] Specifically, identical primers (SEQ ID NO: 1 and 2) were designed for the same Listeria monocytogenes genome (SEQ ID NO: 1) target sequence according to the methods described above. Primers and Listeria monocytogenes genomic material were obtained according to the methods mentioned above and mixed with other PCR reactants to form a 10 μL amplification mixture, the composition of which is shown in Table 5 below. Furthermore, this study set up a series of mixtures containing different initial concentrations (1.0 × 10⁻⁶). -11 M, 1.0 × 10 -12 M, 1.0 × 10 -13 M, 1.0 × 10 -14 M, 1.0 × 10 -15 M, 1.0 × 10 -16 M, 1.0 × 10 -17 M or 1.0×10 -18 The reaction mixture of a 50 bp synthetic Listeria monocytogenes genome sequence fragment (M) was used to compare the rate and sensitivity of the two methods in amplifying and detecting trace amounts of target nucleic acid in samples. A negative control (NTC) was also set up, using an equal volume of water instead of the Listeria monocytogenes genome material and all other components identical.

[0275] Table 5: Composition of the amplification reaction mixture used for sensitivity detection

[0276]

[0277]

[0278] As mentioned above Example 1As described, the amplification reaction system was rapidly thermally cycled between 76°C and 62°C using the CFX Connect™ Real-Time PCR System (Bio-Rad, CA) to perform a rapid SEA reaction. The specific steps are as follows: each thermal cycle consists of the following steps: incubating the amplification mixture at 76°C for 1 second, then immediately lowering the temperature to 62°C, incubating at 62°C for 1 second, and then immediately raising the temperature to 76°C. To achieve real-time monitoring of the amplification process, the fluorescence signal of the amplification reaction mixture was scanned every two thermal cycles, and a fluorescence curve was plotted as a function of time. Figure 7A (1.0×10) -11 M, 1.0 × 10 - 12 M, 1.0 × 10 -13 M and 1.0×10 -14 Data for sample M is not listed.

[0279] In addition, to verify that the observed increase in fluorescence signal corresponds to specific amplification, after the reaction was completed, the amplification mixture was loaded onto a 12.5% ​​polyacrylamide gel for electrophoresis to examine the amplification products. Figure 7B This is a photo of a PAGE gel, showing a concentration of 1.0 × 10⁻⁶. -15 M, 1.0 × 10 -16 M, 1.0 × 10 -17 M and 1.0×10 -18 The accelerated SEA reaction mixture of the original target M produced the expected amplified fragment (43 bp in length), and the negative reaction (NTC) did not show the target band. Lane M is a gradient molecular weight DNA ladder, and the corresponding bands for DNA fragments of 20 bp and 40 bp are marked in the figure.

[0280] The amplification mixture was incubated at 62°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA) to perform an isothermal SEA reaction. To enable real-time monitoring of the amplification process, the fluorescence signal of the amplification products was scanned every minute, and fluorescence curves over time were plotted. Figure 7C 1.0×10 -16 M, 1.0 × 10 -17 M and 1.0×10 -18 Data for sample M is not listed.

[0281] like Figure 7A and 7CAs shown, the fluorescence signals of both methods exhibit a good linear relationship with the initial target concentration in the amplification reaction system. That is, the greater the amount of target present in the initial sample, the less time it takes for the method to generate detectable target molecule amplification products. Notably, in terms of detection rate and sensitivity, the rapid SEA method (under rapid thermal cycling conditions) significantly outperforms the isothermal SEA method (under constant temperature conditions).

[0282] Specifically, such as Figure 7C As shown, the target concentration is 1.0 × 10⁻⁶. -15 At time M, the isothermal SEA method takes approximately one hour to produce detectable amplification products, while... Figure 7A As shown, the rapid SEA method can generate detectable amplification products of the test target under all concentration conditions within 15 minutes. Therefore, compared with the isothermal SEA method, the rapid SEA method reduces the detection time by approximately 75%, thereby shortening the detection time to 15 minutes.

[0283] In addition, such as Figure 7C As shown, within a 20-minute reaction time, the isothermal SEA method was able to detect 1.0 × 10⁻⁶ ppm in the sample. -12 M or higher concentrations of target molecules, and such Figure 7A As shown, the rapid SEA method can detect concentrations as low as 1.0 × 10⁻⁶. -18 The target molecule of M (the target nucleic acid in the sample at this concentration is only a few copies). Therefore, for a reaction time of 15 to 20 minutes, the rapid SEA method improves the detection sensitivity by at least 10%. 6 times.

[0284] 5.8 Example 7: Primer Design

[0285] Primers were designed and evaluated using the NUPACK web tool (www.nupack.org), DNAMelt Web (http: / / unafold.rna.albany.edu / ?q=DINAMelt), NOVOPRO (www.novopro.cn / tools / rev_comp.html), and the BLAST algorithm on the NCBI website (www.ncbi.nlm.nih.gov / tools / primer-blast).

[0286] DNA primers were synthesized by Personal Biotechnology Co., Ltd. (Shanghai, China). The SEA detection kit was purchased from Navid Biotechnology Co., Ltd. (Qingdao, China). The DNA extraction kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). All other reagents and buffers were of analytical grade.

[0287] Traditional PCR reaction Genomic DNA was extracted from Mycoplasma pneumoniae, Chlamydia trachomatis, pork, Bacillus cereus, and Staphylococcus aureus using the TIANamp DNA Extraction Kit (Beijing Tiangen Biotech Co., Ltd., Beijing) according to the manufacturer's instructions. CFX Connect was used. TM Real-time quantitative PCR was performed using a real-time PCR system (Bio-Rad, California, USA). The reaction mixture was 50 μL, containing 20 ng of genomic DNA template, 1 μL of forward and reverse primers (10 μM), 1.5 μL of dNTPs (2.5 mM), 0.25 μL of Taq polymerase, and 5 μL of standard Taq reaction buffer. The reaction procedure included denaturation at 94 °C for 5 min, amplification through 35 thermal cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s), and a final extension at 72 °C for 10 min.

[0288] SEA reaction The SEA reaction was performed in a 10 μL system containing 1 μL template, 1.5 μL each of the two primers (10 μM), 5 μL 2× reaction buffer, and 0.25× Eva Green. To eliminate the influence of extracted genomic DNA purity, unless otherwise specified, the PCR product of the target sequence (1 pM) was used as the template. The reaction mixture was incubated at 57°C, 59°C, 61°C, 63°C, and 65°C for 60 minutes each, and the reaction was controlled via CFX Connect. TM A real-time PCR system (Bio-Rad, California, USA) detected the fluorescence signal of the amplified products every minute. Additionally, a target-free reaction mixture (NTC) was used as a negative control.

[0289] 5.7.1 Optimization of reaction temperature and primer Tm value

[0290] The following examples provide exemplary methods for selecting the optimal reaction temperature and suitable primers for a given polymerase condition.

[0291] The specific steps are as follows: Multiple primers (Mp1-Mp5) specifically for the Mycoplasma pneumoniae 16S rRNA encoding gene with different Tm values ​​(65℃, 63℃, 61℃, 59℃, or 57℃) were designed and synthesized (Table 6). A series of SEA reactions were performed using Bst 2.0 WarmStart DNA polymerase under isothermal conditions of 57℃, 59℃, 61℃, 63℃, or 65℃. To achieve real-time monitoring of the amplification process, the fluorescence signal of the amplification mixture was scanned once per second, and a fluorescence curve was plotted as a function of time. Figure 10 ).

[0292] Table 6. Mycoplasma pneumoniae * 16S rRNA encoding gene-specific primers

[0293]

[0294] * GenBank accession number: CP017343.1

[0295] like Figure 10 As shown, the shortest time (threshold time Tt) for the primer pairs Mp1-Mp5 to produce detectable amplification products in the reaction was 22 min, 15 min, 11 min, 23 min, and 20 min, respectively. Furthermore, among the five reaction temperatures tested, the reaction temperatures at which the primer pairs Mp1-Mp5 reached their minimum Tt values ​​were 61 °C, 61 °C, 61 °C, 61 °C, and 57 °C, respectively. The observed results are summarized in Table 6 above.

[0296] These results indicate that when using Bst DNA polymerase for SEA or rapid SEA reactions, primers with a reaction temperature of approximately 61°C and a Tm value of approximately 61°C can be preferentially selected and used.

[0297] Subsequently, to demonstrate that the optimal conditions (including reaction temperature and primer properties) determined by the above method are applicable to practical applications, these optimal conditions were applied to SEA reactions targeting Mycoplasma pneumoniae genomic DNA (different from the synthesized and / or purified DNA fragments used in research laboratories). The specific steps were as follows: 40 ng of Mycoplasma pneumoniae genomic DNA was used as a template for the SEA reaction with primer pair Mp3 at the same reaction temperatures (i.e., 65°C, 63°C, 61°C, 59°C, or 57°C). Similar results were observed: the reaction using primer pair Mp3 at 61°C showed the shortest Tt value. Although the shortest Tt value in this reaction (approximately 20 minutes) was longer than the Tt value in the reaction using the amplified target DNA fragment as the target, this difference can be attributed to the lower probability of denaturation vesicles appearing at the target site in longer genomic nucleic acids compared to shorter target DNA fragments. Figure 10 F). These results further demonstrate that the steps and methods of this embodiment can be used to determine the optimal reaction temperature and primer Tm value for the SEA method and current accelerated SEA methods.

[0298] In the aforementioned studies on optimizing primer Tm values ​​and reaction temperatures, it was observed that the Tt value is also related to the difference in Tm values ​​between the two primers in a primer pair. The following examples provide other exemplary methods for selecting primers with favorable Tm value characteristics.

[0299] Specifically, primer pairs specific to Chlamydia trachomatis (Ct1-Ct3) or domestic pigs (Sd1-Sd3) with different Tm values ​​were designed and tested in SEA reactions at 61℃ (Table 7). The average Tm values ​​of the primer pairs were all close to 61℃ to rule out the possible influence of this factor. Figure 11 As shown, for both Chlamydia trachomatis and domestic pig-specific primers, the primer pairs with the smallest difference in Tm values ​​had the shortest Tt values, while the primer pairs with the largest difference in Tm values ​​showed the highest Tt values. Primer pairs with similar Tm values ​​usually have similar annealing temperatures, and therefore similar amplification reaction rates, resulting in higher efficiency of the SEA reaction (Thornton et al., “Real-time PCR (qPCR) primer design using free online software,” Biochem. Mol. Biol. Edu., (2011) 39: 145-154). These results suggest that primer pairs with similar Tm values ​​can be preferentially selected for SEA reactions and for accelerating SEA reactions.

[0300] Table 7. Chlamydia trachomatis (C. trachoma) * )16S rRNA encoding gene and domestic pig (S. domesticica ** 18S rRNA encoding gene-specific SEA primers

[0301]

[0302] * GenBank ID: NR_025888.1

[0303] ** GenBank accession number: JN601073.1

[0304] 5.7.2 Optimization of 3' end G / C content

[0305] The following examples provide an exemplary method for optimizing the G / C content of primers in conjunction with this method.

[0306] Specifically, SEA reactions were performed using primer pairs specific to the target sequences of the encoding genes of Mycoplasma pneumoniae 16S rRNA (Mp3, Mp6, and Mp7) or Chlamydia trachomatis 16S rRNA (Ct1, Ct4, and Ct5). The polymerase selected in this example was Bst DNA polymerase. The specific steps were as follows: Mycoplasma pneumoniae-specific primers were designed such that the total number of G and C in the 5nt region at the 3' end was 1 to 4. Chlamydia trachomatis-specific primers were designed such that the total number of G and C in the 5nt region at the 3' end was 2 or 3. Furthermore, the mean Tm value of all primer pairs was around 61°C, and the reaction was performed at a constant temperature of 61°C. To monitor amplification in real time, the fluorescence signal of the amplification mixture was scanned once per second, and a fluorescence curve was plotted over time. Figure 12 The number of G / Cs in the 3' end of the primers, the number of G / Cs in the 5nt region at the 3' end of each primer pair, and the Tt value of the reaction are shown in Table 8.

[0307] Table 8. Mycoplasma pneumoniae * ) and Chlamydia trachomatis (C. trachoma ** 16S rRNA encoding gene-specific SEA primers

[0308]

[0309] * GenBank accession number: CP017343.1

[0310] ** GenBank ID: NR_025888.1

[0311] As above Figure 11 As shown in A and Table 8, the results related to Mycoplasma pneumoniae-specific primers indicate that primers with higher G / C content near the 3' end exhibit lower Tt values, suggesting that primers with higher G / C content at the 3' end can be preferentially selected. This observation contradicts the design principles of conventional PCR primers, which generally avoid primers with high G / C content at the 3' end (Simonsson et al., “DNA tetraplex formation in the control region of c-myc,” Nucleic Acids Res., (1998) 26:1167-1172).

[0312] Furthermore, it was observed that although the G / C content in the 3' terminal regions of the three Chlamydia trachomatis-specific primer pairs was similar, the primer pair with all 3'-terminal nucleotides being either G or C (Ct1) had the lowest Tt value. The same phenomenon was observed in the results for Mycoplasma pneumoniae-specific primers. These results indicate that the G / C base pairing between the primer tail and its target site is relatively more stable and more conducive to hybridization because it avoids the primer being easily replaced by the original complementary strand. In addition, the stable structure formed by the terminal base pair will facilitate primer extension initiated by polymerase and prevent non-specific amplification (Rodríguez-Lázaro et al., “Real-time PCR in food science: introduction,” Curr. Issues Mol. Biol (2013) 15:25-38).

[0313] Therefore, this study demonstrates that the primers used in this invention should have at least two Gs and / or Cs in the 5-nt region at the end where the polymerase intends to extend. Furthermore, primers with Gs or Cs at their ends are more conducive to polymerase-initiated extension.

[0314] 5.7.3 Primer sequence optimization based on complementarity

[0315] The following examples provide exemplary procedures for optimizing primer sequences to avoid or reduce the formation of self-complementary secondary structures within primer molecules.

[0316] The effect of self-complementation or 3' complementarity between primers in primer pairs was evaluated using the SEA method. Specifically, the number of potential self-complementary or 3' complementary sites was analyzed for different primer pairs specific to Chlamydia trachomatis (Ct1, Ct6, and Ct2) or Bacillus cereus (Bc1-Bc3). The BLAST algorithm from the NCBI website (www.ncbi.nlm.nih.gov / tools / primer-blast) was used. The predicted number of complementary sites for each primer is summarized in Table 9. The primers were then subjected to the SEA reaction under the above conditions. To monitor amplification in real time, the fluorescence signal of the amplification mixture was scanned once per second, and fluorescence curves were plotted over time. Figure 13 ).

[0317] Table 9. Chlamydia trachomatis (C. trachoma) * ) and Bacillus cereus ** 16S rRNA encoding gene-specific SEA primers

[0318]

[0319] *GenBank ID: NR_025888.1

[0320] ** GenBank ID: NR_152692.1

[0321] like Figure 13 As shown, the number of complementary sites in primer pairs is positively correlated with the Tt value of the corresponding reaction, with the primer pair having the fewest potential complementary sites having the lowest Tt value. Furthermore, it was observed that among Bacillus cereus-specific primers, the primer pair with the lowest Tt value (Bc1) had the highest Tm value (65℃). Additionally, the 3' terminal nucleotide of the Bc1 P2 primer was neither G nor C. This finding indicates that intra- and inter-sequence complementarity negatively impacts the overall efficiency and rate of the SEA or rapid SEA method, outweighing the positive effects of appropriate primer G / C content or Tm value.

[0322] Therefore, this study shows that avoiding or reducing potential self-complementation and / or 3' complementarity in primer sequences is beneficial to improving the efficiency of this method.

[0323] 5.7.4 Priority considerations for primer design (Tm value and 3' end C / G content)

[0324] In actual primer design, considering various primer optimization considerations can lead to conflicting primer selections. For example, ... Figure 13 As shown in Table 9, the negative impacts of intra- and inter-sequence complementarity outweigh the positive impacts of appropriate primer G / C content or Tm value on overall efficiency and amplification rate. The following studies further provide exemplary procedures and steps for determining the priority between primer Tm value and 3' end G / C content.

[0325] Specifically, 4 ng of genomic DNA was used as a template, and an SEA reaction was performed using two primer pairs specific to Staphylococcus aureus (Sa1 and Sa2). For the Sa1 primer pair, the Tm values ​​and Tm differences between the two primers were approximately 65 °C and 2.2 °C, respectively. For the Sa2 primer pair, the Tm values ​​and Tm differences between the two primers were approximately 61 °C and 1.1 °C, respectively, and both primers had A or T nucleotides at their 3' ends. The primer sequences and characteristics are summarized in Table 10 below. The primers were used for the SEA reaction under the above conditions. To monitor the amplification in real time, the fluorescence signal of the amplification mixture was scanned once per second, and a fluorescence curve was plotted as a function of time. Figure 14 ).

[0326] Table 10. Staphylococcus aureus * 16S rRNA encoding gene-specific primers

[0327]

[0328] * GenBank accession number: D83356.1.

[0329] like Figure 14 As shown in Table 10, the amplification efficiency of primer pair Sa1 (Tt: 37 min) was significantly lower than that of primer pair Sa2 (Tt: 32 min). Based on these measurements, it can be concluded that choosing a favorable Tm value and the Tm value difference is of higher priority than choosing a favorable 3' end residue or 3' end G / C content. These observations can be interpreted as an effective promotion of stable primer-target double-stranded structures between primers with appropriate Tm values ​​and reaction temperatures, compared to the stability provided by GC base pairing.

[0330] In summary, these studies indicate that, based on different considerations, the priority order for primer design is (from high to low priority): (1) avoiding / reducing self-complementarity and / or 3' complementarity in the primer sequence, (2) selecting appropriate Tm values ​​and / or Tm value differences, and (3) selecting appropriate C / G content and / or G / C as terminal residues in the 3' 5nt region. In other words, when the selection of primer sequences based on lower priority considerations contradicts the selection of primer sequences based on higher priority considerations, the selection based on higher priority considerations can be adopted.

[0331] 5.9 Example 8: Reagent Kit

[0332] The following provides examples of detecting target nucleic acids using the rapid SEA method of the present invention with a pre-prepared kit.

[0333] A kit containing buffer A and buffer B with the following components was prepared.

[0334] Buffer A:

[0335] Isothermal reaction buffer (10×): 1.75 μL;

[0336] dNTPs (10 mM): 2 μL;

[0337] Primer 1: 7.5 μL (final concentration: 3.0 × 10⁻⁶) -6 M);

[0338] Primer 2: 7.5 μL (final concentration: 3.0 × 10⁻⁶) -6 M);

[0339] Polyethylene glycol (PEG) 200 (100%): 0.625 μL;

[0340] Evagreen (20×): 0.625 μL;

[0341] Buffer B:

[0342] Isothermal reaction buffer (10×): 0.75 μL;

[0343] ET single-chain binding protein (SSB) (500 μg / mL): 0.25 μL;

[0344] DNA polymerase (8 U / μL): 0.75 μL.

[0345] In this embodiment, primer pairs were designed to detect Staphylococcus aureus in the sample. Specifically, primers were designed to amplify a fragment of the Staphylococcus aureus 16S rRNA encoding gene, with the following sequence: 5'-GGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGC-3' (SEQ ID NO:4).

[0346] The primer sequences are:

[0347] Primer 1: 5'-GGTTCAAAAGTGAAAGACGGTCTTG-3' (SEQ ID NO: 5);

[0348] Primer 2: 5'-GCGCGGATCCATCTATAAGTGAC-3' (SEQ ID NO: 6).

[0349] According to the manufacturer's instructions, Staphylococcus aureus genome was extracted using a DNA / RNA isolation kit purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China, catalog number DP422). Three parallel replicates were prepared as follows: Mixing buffer A and buffer B were prepared, and 2.5 μL of the extracted Staphylococcus aureus genome material was added to the mixture, followed by water to a total volume of 25 μL. An amplification mixture was prepared using the same amount of water instead of the Staphylococcus aureus genome material as a negative control (NTC).

[0350] Using CFX Connect TM The real-time PCR system (Bio-Rad, CA) enables rapid SEA reactions by subjecting the amplification reaction system to rapid thermal cycling from 76°C to 61°C. Specifically, each thermal cycle consists of the following steps: incubating the amplification reaction system at 76°C for 1 second, then immediately lowering the temperature to 62°C, incubating at 62°C for 1 second, and then immediately raising the temperature back to the previous 76°C. To achieve real-time monitoring of the amplification process, the fluorescence signal of the amplification products is scanned every two thermal cycles, and a fluorescence curve over time is plotted. Figure 8As shown in the figure, the amplification results of the three replicate experiments were consistent, while the negative control did not produce a detectable fluorescence signal. These results indicate that reproducible and stable results can be obtained using this kit, and that the reagents stored in buffer A and buffer B respectively are stable and can induce a reaction when mixed.

[0351] 5.10 Microfluidic devices

[0352] The following provides an example of using the rapid SEA method of the present invention to detect target nucleic acids in a microfluidic chip.

[0353] A 10 μL reaction mixture containing the following substances was prepared:

[0354] Purified water: 0.35μL

[0355] Isothermal reaction buffer (10×): 1 μL

[0356] Primer 1: 3 μL (final concentration: 3.0 × 10⁻⁶) -6 M);

[0357] Primer 2: 3 μL (final concentration: 3.0 × 10⁻⁶) -6 M);

[0358] PEG 200 (100%): 0.25 μL;

[0359] Evagreen (20×): 0.25 μL;

[0360] dUTP (10mM): 0.8μL;

[0361] Uracil DNA glycosylation enzyme (1 U / μL): 0.1 μL;

[0362] DNA polymerase (8 U / μL): 0.25 μL;

[0363] Target nucleic acid: 1 μL.

[0364] In this embodiment, primer pairs were designed to detect Staphylococcus aureus in the sample. Specifically, primers were designed to amplify a fragment of the Staphylococcus aureus 16S rRNA encoding gene, the sequence of which is as follows:

[0365] 5'-GGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGC-3' (SEQ ID NO: 4)

[0366] The primer sequences are:

[0367] Primer 1: 5'-GGTTCAAAAGTGAAAGACGGTCTTG-3' (SEQ ID NO: 5);

[0368] and

[0369] Primer 2: 5'-GCGCGGATCCATCTATAAGTGAC-3' (SEQ ID NO: 6).

[0370] According to the manufacturer's instructions, the DNA / RNA isolation kit purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China, catalog number DP422) was used to extract Staphylococcus aureus genome into a stock solution. All reagents were mixed, and 1.0 μL of different concentrations of Staphylococcus aureus target was added to the mixture to bring the total volume to 10 μL. The target concentrations were 1.0 × 10⁻⁶. -9 M, 1.0 × 10 -10 M, 1.0 × 10 -11 M, 1.0 × 10 -12 M, 1.0 × 10 -13 M, 1.0 × 10 -14 M and 1.0×10 - 15 M. The reaction also included a negative control (NTC) mixture with an equal volume of water instead of the Staphylococcus aureus genomic material and all other components identical.

[0371] The reaction mixture was thoroughly mixed, and then all the mixture was pipetted into the reaction chambers of the microfluidic chip Rapi:chip™ (Genesystem, Korea), each with an inlet and an vent. After all the sample and NTC mixtures were injected into the reaction chambers, a sealing film was affixed to the microfluidic chip to seal the inlet and vent. The microfluidic chip was then placed in a UF-150 GENECHECKER. TM On an ultra-fast real-time PCR system (Genesystem, South Korea), preparations are underway for an amplification reaction.

[0372] After incubation at 37°C for 5 minutes, the microfluidic chip underwent rapid thermal cycling between 76°C and 60°C. Each thermal cycle consisted of the following steps: incubation of the amplification mixture at 76°C for 1 second, followed immediately by cooling to 60°C, incubation at 60°C for another 1 second, and then immediately warming back to 76°C. The rate of temperature increase and decrease was 8°C / s, and each cycle was completed within 12 seconds. To monitor amplification in real time, the fluorescence signal of the amplification product was scanned once after each thermal cycle, and a fluorescence curve was plotted as a function of time. Figure 15 As shown in the figure, the appearance time of the fluorescence signal is positively correlated with the concentration. The negative control, however, did not produce a detectable fluorescence signal.

[0373] The study in this embodiment demonstrates that the rapid SEA method of the present invention can amplify and detect target molecules (concentrations as low as 1.0 × 10⁻⁶) in a sample within 8 minutes (less than 40 cycles). -14 M or approximately 6.0 × 10⁻⁶ in a 10 μL reaction system. 4 One copy.

[0374] 5.11 Example 10: Uracil-DNA Glycosyltransferase (UDG) Reduces Contamination

[0375] The following studies demonstrate that adding uracil-DNA glycosylation enzyme to the amplification mixture can reduce residual contamination during amplification.

[0376] First, the following studies were conducted to demonstrate that rapid SEA can synthesize novel amplification products using dUTPs. In the first reaction (dTTPs; solid circles), the amplification mixture contained dNTPs (dATPs, dGTPs, dTTPs, dCTPs), and in the second reaction (dUTPs; solid triangles), the amplification mixture contained dNTPs (dATPs, dGTPs, dUTPs, dCTPs). A control reaction (NTC; solid squares) without the target molecule was also included. The above reaction mixtures were subjected to rapid SEA, and fluorescence curves were plotted over time. Figure 16 As shown in the figure, replacing dTTPs with dUTPs does not significantly affect the reaction efficiency, indicating that dUTPs can be used for fast SEA reactions.

[0377] Furthermore, the following study was conducted to demonstrate the digestion of uracil-containing nucleic acids by UDG. The uracil-containing amplification product (using dUTPs instead of dTTPs) from the second reaction described above was digested with UDG. Specifically, UDG (0.01 U / μL) was added to 10 μL of the amplification mixture to digest the amplification product, and the mixture was incubated at 37°C for 2 minutes. The digested product was then loaded onto an SDS gel for electrophoresis (lane 2). A 10 μL amplification mixture without UDG treatment was loaded onto another lane of the SDS gel (lane 1) for comparison. Figure 17 As shown, the fluorescence of the amplified product after UDG treatment was significantly weaker than that of the untreated amplified product, indicating that UDG degraded the uracil-containing amplified product by cleaving the U base incorporated into the product.

[0378] Finally, the following studies were conducted to demonstrate that adding UDG to the amplification mixture during rapid SEA can effectively prevent contamination caused by residual amplification products (e.g., aerosols) present in the surrounding environment.

[0379] In this study, dATPs, dGTPs, dUTPs, and dCTPs were used for the first round of rapid SEA reactions. The amplified products were then used as targets for a second round of rapid SEA reactions. Specifically, rapid SEA reactions were performed as described above, and fluorescence curves depicting the fluorescence signal over time for the second round of reactions were plotted. Figure 18 As can be seen from the figure, the amplification reaction threshold time (Tt) with 0.01 U / μL UDG was delayed by 3.78 minutes compared to the amplification reaction without UDG (solid square), indicating that UDG can effectively prevent nucleic acid molecule contamination in the current rapid SEA method.

[0380] 5.12 Example 11: Rapid amplification and detection of DNA molecules in samples using thermostable Taq DNA polymerase.

[0381] The following studies were conducted to examine the ability of the rapid SEA method to detect DNA molecules in biological samples.

[0382] Based on the target nucleic acid sequence, a pair of specific primers was designed using NUPACK software (www.nupack.org / ). Specifically, the target sequence is...

[0383] 5'-AGATGTTGAAGGATTCAACCAAATCTCCAGAGTTTGTTAAAACCGTTCCAA-3'(SEQ ID NO:58),

[0384] The primer sequences are:

[0385] Primer 1: 5'-ATGTTGAAGGATTCAACCAAATC-3' (SEQ ID NO: 59);

[0386] Primer 2: 5'-GGAACGGTTTTAACAAACTCTG-3' (SEQ ID NO: 60).

[0387] Primers and target DNA molecules were commercially synthesized (Sangon Biotech, Shanghai, China) and mixed with other PCR reactants to form 10 μL amplification mixtures, as shown in Table 11 below. Specifically, amplification mixtures were prepared, each containing 1.0 × 10⁻⁶ molecules. -12 M, 1.0 × 10 -13 M, 1.0 × 10⁻ 14 M, 1.0 × 10 -15 M or 1.0×10 -16 M. parahaemolyticus genomic material. Primer concentration was 5.0 × 10⁻⁶. -7M, with a polymerase concentration of 0.05 U / μL, and including an amplification mixture with an equal volume of water replacing the target DNA as a negative control (NTC).

[0388] Table 11: Content of Amplification Mixtures Used for DNA Amplification

[0389]

[0390] To perform a rapid SEA reaction, the amplification mixture was rapidly thermally cycled between 76°C and 61°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA). Specifically, each thermal cycle consisted of incubating the amplification reaction at 76°C for 1 second, immediately followed by a temperature reduction to 61°C, incubation at 61°C for 1 second, and then immediately returning to the previous temperature of 76°C. To enable real-time monitoring of the amplification process, the fluorescence signal of the amplification products was scanned every two thermal cycles, and fluorescence curves over time were plotted. Figure 19 ).

[0391] As shown in the figure, the rapid SEA method can effectively detect the genomic nucleic acid of Vibrio parahaemolyticus at the provided target concentration, which enables the use of the method and kit of this invention for immediate diagnosis of pathogen infection.

[0392] 5.13 Example 12: Amplification and detection of DNA molecules in a sample using nucleic acid probes.

[0393] The following study was conducted to examine the ability of a rapid SEA method using nucleic acid probes to detect DNA molecules in biological samples.

[0394] Specifically, a pair of specific primers and probes were designed based on the following target sequences in the human β-actin gene:

[0395] 5'-CAAATGCTTCTAGGCGGACTATGACTTAGTTGCGTTACACCCTTTCTTGACAAAACCTAACTTGCG-3'(SEQ ID NO:61)

[0396] The primer and probe sequences are as follows:

[0397] Primer 1: 5'-CCTGTGTTATCTTGGAGGTC-3' (SEQ ID NO: 62);

[0398] Primer 2: 5'-FAM-CCCTGAAGGGCTCTCTGG-BHQ-3' (SEQ ID NO: 63).

[0399] Probe: 5'-ACCAAAAGAGCTAGAACCAC-3' (SEQ ID NO: 64).

[0400] Primers, probes, and target DNA molecules were commercially synthesized (Sangon Biotech, Shanghai, China) and mixed with other PCR reactants to form a 10 μL amplification mixture, as shown in Table 12 below. Specifically, the amplification mixture contained genomic material isolated from human oral epithelial cells as target DNA. The primer concentration was 5.0 × 10⁻⁶. -7 M, probe concentration is 6.0 × 10 -7 M, with a polymerase concentration of 0.05 U / μL, and including an amplification mixture with an equal volume of water replacing the target DNA as a negative control (NTC).

[0401] Table 12: Content of amplification mixture used for DNA amplification

[0402] Element Final concentration Primer 1 <![CDATA[5.0×10 -7 M]]> Primer 2 <![CDATA[5.0×10 -7 M]]> probe <![CDATA[6.0×10 -7 M]]> dNTPs 8mM target RNA <![CDATA[1.0×10 -14 M synthesized DNA fragment]]> Taq reaction buffer 1× Taq DNA polymerase 0.05 U / μL 100% Polyethylene Glycol 200 0.625μL pure water Add to 10μL

[0403] To perform a rapid SEA reaction, the amplification mixture was rapidly thermally cycled between 76°C and 61°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA). Specifically, each thermal cycle consisted of incubating the amplification reaction at 76°C for 1 second, immediately followed by a temperature reduction to 61°C, incubation at 61°C for 1 second, and then immediately returning to the previous temperature of 76°C. To enable real-time monitoring of the amplification process, the fluorescence signal of the amplification products was scanned every two thermal cycles, and fluorescence curves over time were plotted. Figure 20 ).

[0404] As shown in the figure, the rapid SEA method can efficiently detect the human β-actin gene from oral epithelial cells within approximately 10 minutes at the provided target concentration. In three replicate reactions, amplification reached the exponential phase at approximately the same time, while no amplification product was detected in the negative control group, indicating that the method and reaction system have high reproducibility and stability.

[0405] 5.14 Example 13: Amplification and Detection of DNA Molecules in Samples

[0406] The following study was conducted to examine the ability of the rapid SEA method to detect DNA molecules in biological samples.

[0407] A pair of specific primers was designed based on the target nucleic acid sequence using NUPACK software (www.nupack.org / ). Specifically, the target sequence is...

[0408] 5'-AGATGTTGAAGGATTCAACCAAATCTCCAGAGTTTGTTAAAACCGTTCCAA-3'(SEQ ID NO:58),

[0409] The primer sequences are:

[0410] Primer 1: 5'-ATGTTGAAGGATTCAACCA-3'(M58822.1 b)(SEQ ID NO:65);

[0411] Primer 2: 5'-GGAACGGTTTTAACAAACT-3'(M58822.1 b) (SEQ ID NO: 66).

[0412] Primers and target DNA molecules were commercially synthesized (Sangon Biotech, Shanghai, China) and mixed with other PCR reactants to form a 10 μL amplification mixture, as shown in Table 13 below. Specifically, the amplification mixture was prepared containing 1.0 × 10⁻⁶ cells of Listeria monocytogenes. -12 M synthesizes the target DNA fragment. Primer concentration is 3.0 × 10⁻⁶. -6 M, with a polymerase concentration of 0.24 U / μL, and including an amplification mixture with an equal volume of water replacing the target DNA as a negative control (NTC).

[0413] Table 13: Content of Amplification Mixtures Used for DNA Amplification

[0414] Element Final concentration Primer 1 <![CDATA[3.0×10 -6 M]]> Primer 2 <![CDATA[3.0×10 -6 M]]> target DNA <![CDATA[1.0×10 -12 M synthesized DNA fragment]]> dNTPs 8mM Isothermal reaction buffer 1× 20×Evagreen 0.625μL ET SSB 5μg / mL Bst DNA polymerase 0.24 U / μL 100% Polyethylene Glycol 200 0.625μL pure water Add to 10μL

[0415] To perform a rapid SEA reaction, the amplification mixture was rapidly thermally cycled between 76°C and 55°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA). Specifically, each thermal cycle consisted of incubating the amplification reaction at 76°C for 1 second, immediately followed by a temperature reduction to 55°C, incubation at 55°C for 3 seconds, and then immediately returning to the previous temperature of 76°C. To enable real-time monitoring of the amplification process, the fluorescence signal of the amplification products was scanned every two thermal cycles, and fluorescence curves over time were plotted. Figure 21 ).

[0416] As shown in the figure, the rapid SEA method can effectively detect Listeria monocytogenes synthetic DNA fragments in less than 10 minutes at the provided target concentration, which enables on-site, real-time detection of pathogen infection using the method and kit of this invention.

[0417] 5.15 Example 14: Amplification and Detection of Small RNA Molecules in Samples

[0418] The following study was conducted to examine the ability of a rapid SEA method to detect small RNA molecules in biological samples. A pair of specific primers was designed based on the target nucleic acid sequence using NUPACK software (www.nupack.org / ). Specifically, the target sequence was...

[0419] 5'-GCUUAUCAGACUGAUGUUGA-3'(SEQ ID NO:67),

[0420] The primer sequences are:

[0421] Primer 1: 5'-GCTTATCAGA-3'(M58822.1 b)(SEQ ID NO:68);

[0422] Primer 2: 5'-TCAACATCAG-3'(M58822.1 b)(SEQ ID NO:69).

[0423] Primers and target DNA molecules were commercially synthesized (Sangon Biotech, Shanghai, China) and mixed with other PCR reactants to form a 10 μL amplification mixture, as shown in Table 14 below. Specifically, the amplification mixture was prepared containing 1.0 × 10⁻⁶ molecules. -11 The target small RNA fragment for M synthesis. Primer concentration: 3.0 × 10⁻⁶ -6 M, polymerase concentration was 0.25 U / μL. An amplification mixture containing an equal volume of water replacing the target small RNA fragment was included as a negative control (NTC).

[0424] Table 14: Content of amplification mixture used for small RNA amplification

[0425] Element Final concentration Primer 1 <![CDATA[3.0×10 -6 M]]> Primer 2 <![CDATA[3.0×10 -6 M]]> target small RNA <![CDATA[1.0×10 -11 M <!-- 63 -->]]> dNTPs 8mM Isothermal reaction buffer 1× 20×Evagreen 1.25μL Klenow clips exo- 0.25 U / μL Uracil-DNA glycosylation enzyme 0.05 U / μL 100% Polyethylene Glycol 200 1.25μL pure water Add to 20μL

[0426] To perform a rapid SEA reaction, the amplification mixture was rapidly thermally cycled between 60°C and 34°C using a CFX Connect™ real-time PCR system (Bio-Rad, CA). Specifically, each thermal cycle consisted of incubating the amplification reaction at 60°C for 1 second, immediately followed by a temperature reduction to 34°C, incubation at 34°C for 1 second, and then immediately returning the temperature to the previous 60°C. To enable real-time monitoring of the amplification process, the fluorescence signal of the amplification products was scanned every two thermal cycles, and fluorescence curves over time were plotted. Figure 22 ).

[0427] As shown in the figure, the rapid SEA method can effectively detect synthetic small RNA fragments in less than 10 minutes at the provided target concentration, which makes it possible to detect small RNAs from samples using this method and kit.

[0428] 6. Sequence List

[0429] This application was filed with a computer-readable (CRF) copy of the sequence list named 14624-002-146_ST25.TXT, created in xxx, 2020, and measuring 11,518 bytes; the entire contents of which are incorporated herein by reference. sequence list <110> Qingdao University <120> Methods and kits for amplifying and detecting nucleic acids <130> 14624-002-888 <140> TBA <141> <160> 69 <170> PatentIn version 3.5 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 1 gggtcattgg aaactggaag actggagtgc agaagaggag agtggaattc 50 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 2 gtcattggaa actggaagac tg 22 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 3 ccactctcct cttctgcac 19 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 4 ggttcaaaag tgaaagacgg tcttgctgtc acttatagat ggatccgcgc 50 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 5 ggttcaaaag tgaaagacgg tcttg 25 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 6 gcgcggatcc atctataagt gac 23 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primers (SEQ ID Nos. 2 and 3) were redesigned to test the rapid SEA method. The ability to detect RNA molecules in biological samples <400> 7 gggtcauugg aaacuggaag acuggagugc agaagaggag aguggaauuc 50 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 8 tcgcggtaat acataggtcg c 21 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 9 gcccaataaa tccggataac gc 22 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 10 gtctggtgtt aaaggcagc 19 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 11 tccaatgcat acaactgtta agc 23 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 12 gcaagggttc gttatttgat g 21 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 13 ctagctgata tggcgcac 18 <210> 14 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 14 gctatgctga gaagtagaat ag 22 <210> 15 <211> 18 <212> DNA <2...

Claims

1. A method for amplifying a target nucleic acid molecule in a sample, the method comprising contacting a polymerase and a pair of oligonucleotide primers with the sample to form an amplification mixture, wherein the primers are designed to specifically hybridize with the target nucleic acid molecule; subjecting the amplification mixture to multiple thermal cycles between a first temperature and a second temperature to amplify the target nucleic acid sequence by PCR; wherein the polymerase is wild-type DNA polymerase I, DNA polymerase I large fragment Klenow, or Klenow exo-; The first temperature is in the range of 50-60℃; the second temperature is in the range of 30-40℃. The primers are 10 to 15 nucleotides in length; The amplification product is 20-30 base pairs long; Each thermal cycle includes incubating the amplification mixture at the first temperature for about 1 second and at the second temperature for about 1 second, wherein the temperature change time is less than 2 seconds; The amplification mixture also contains at least 0.5% polyethylene glycol by volume or at least 1 μg / mL SSB.

2. The method according to claim 1, wherein, The sequence length ratio of at least one of the primers to the amplification product is 30-60%.

3. The method according to claim 1, wherein, The G / C content of at least one of the primers is 40% to 60%, and the difference in the percentage of G / C content of the primers is less than 20%.

4. The method according to claim 1, wherein, At least one of the primers has an extension end from which the polymerase can add nucleotides during PCR, and the primer has a G or C at the extension end.

5. The method according to claim 4, wherein, At least one of the primers has an extension end from which the polymerase can add nucleotides during PCR, and wherein the primer has a G / C content of at least 40% in a continuous 5-nucleotide region at the extension end.

6. The method according to claim 1, wherein, The method completes at least 35 thermal cycles in less than 10 minutes, or at least 40 thermal cycles in less than 8 minutes.

7. The method according to claim 1, further comprising detecting the amplified sequence.

8. The method of claim 1, wherein the amplification mixture further comprises dUTPs.

9. The method according to claim 8, wherein, The amplification mixture does not contain dTTPs.

10. The method according to claim 8 or 9, wherein, The amplification mixture also contains uracil-DNA glycosylase UDG.

11. The method of claim 1, wherein the amplification mixture comprises a concentration not exceeding 1.0 × 10⁻⁶. -12 The target nucleic acid of M.

12. The method of claim 1, wherein the amplification mixture comprises fewer than 10 copies of the target nucleic acid.

13. The method according to claim 1, wherein, The amplification mixture contains the polymerase at a concentration of not less than 0.1 U / μL.

14. The method of claim 1, wherein the amplification mixture contains a concentration of not less than 1.0 × 10⁻⁶. -6 At least one primer for M.

15. The method according to claim 1, wherein the volume of the amplification mixture is 1-30 μL.

16. The method according to claim 1, wherein, The processing steps are performed by loading the amplification mixture onto a microfluidic device capable of cooling and heating the amplification mixture at a rate of at least 10 °C / s.

17. The method according to claim 1, wherein the target nucleic acid is a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule.

18. The method of claim 1, wherein the target nucleic acid is DNA or RNA.

19. The method of claim 18, wherein the target nucleic acid is a small RNA.

20. A method for detecting a target nucleic acid molecule in a sample, the method comprising contacting a polymerase and a pair of oligonucleotide primers with the sample to form an amplification mixture, wherein the primers are designed to specifically hybridize with the target nucleic acid molecule; The amplification mixture is subjected to multiple thermal cycles between a first temperature and a second temperature to rapidly amplify the target nucleic acid sequence via PCR, thereby detecting the amplified sequence in the amplification mixture. The polymerase mentioned is wild-type DNA polymerase I, or the large fragment of DNA polymerase I, Klenow or Klenow exo-. The first temperature is in the range of 50-60℃; the second temperature is in the range of 30-40℃. The primers are 10 to 15 nucleotides in length; The amplification product is 20-30 base pairs long; Each thermal cycle includes incubating the amplification mixture at the first temperature for about 1 second and at the second temperature for about 1 second, wherein the temperature change time is less than 2 seconds; The amplification mixture also contains at least 0.5% polyethylene glycol by volume or at least 1 μg / mL SSB.

21. The method according to claim 20, wherein, The detection is performed at intervals of 1, 2, 5, or 10 thermal cycles.

22. The method according to claim 20, wherein, The test is performed by detecting a fluorescence signal in the amplification mixture, which reflects the content of the amplified sequence.

Images