Method for Asymmetric Amplification of Target Nucleic Acid

Abstract

A method for multiplex asymmetric amplification of one or more target nucleic acids in a sample is provided, which can simultaneously amplify multiple target nucleic acids present in the sample and simultaneously generate multiple single-stranded products.

Description

Technical Field

[0001] This application relates to multiplex asymmetric amplification of nucleic acid molecules. In particular, this application provides a method for simultaneously and asymmetrically amplifying one or more target nucleic acids in a sample, which can simultaneously and asymmetrically amplify a plurality of target nucleic acids present in the sample and can simultaneously generate a large amount of single-stranded products.

Background Art

[0002] Asymmetric PCR was first described by Non-Patent Document 1 and refers to a method of generating a large amount of single-stranded DNA (ssDNA) using an unequal amount of primer pairs. The single-stranded DNA generated by asymmetric PCR can be used for sequencing, as a probe, or to improve the detection signal in real-time PCR, microarray detection, and probe melting curve analysis. However, in conventional asymmetric PCR, careful optimization is often required to maximize the production of specific single-stranded products and minimize non-specific amplification. When it is necessary to simultaneously and asymmetrically amplify multiple target nucleic acid sequences, an increase in primer pairs leads to an increase in non-specific amplification such as primer dimers, further increasing the difficulty of design and optimization.

[0003] In conventional asymmetric PCR amplification, due to the decrease in the concentration of limiting primers, their melting point becomes lower than the annealing temperature of the PCR reaction, resulting in inefficient asymmetric PCR amplification. In response to this, Non-Patent Document 2 uses the actual primer concentration as a factor affecting the primer melting point (T m ) value when designing primers, and also the T mIt was expected that [the relevant factor] would increase and become higher than the annealing temperature, and based on this, LATE-PCR (Linear-After-The-Exponential-PCR) was proposed, in which the amplification efficiency of asymmetric PCR is improved and a large amount of single-stranded products are generated. This method solves the problem of low amplification efficiency of asymmetric PCR and facilitates the optimization of asymmetric PCR. However, this method cannot solve the problem that non-specific amplification caused by an increase in the primer pair increases, so it is still difficult to achieve multiplex asymmetric PCR amplification.

[0004] Non-Patent Document 3 describes the Homo-Tag Assisted Non-Dimer System (HAND system). In this system, the same tag sequence is added to the 5'-ends of both the target-specific upstream and downstream primers to form tailed / tagged target-specific primers. In PCR amplification, the first PCR amplification is first initiated by low-concentration tailed / tagged specific primers at a lower annealing temperature. After several cycles, subsequent amplification of the amplification products of the first PCR amplification is carried out using high-concentration universal primers at a high annealing temperature. Since all the specific primers contain the same tag sequence, all the products generated by the first PCR amplification (including primer dimers) have complementary tag sequences at their ends. Due to the high local concentration, the single strands of small fragment products such as primer dimers are prone to self-annealing and form stable "pan-handle" structures, preventing further annealing of the universal primers and thereby inhibiting the amplification of primer dimers. By combining the use of low-concentration homogeneously tagged target-specific primers and high-concentration universal primers, the HAND system can effectively suppress the amplification of primer dimers, achieve efficient multiplex PCR amplification, and maintain high amplification efficiency and detection sensitivity. However, PCR amplification using the HAND system is symmetric amplification and cannot generate single-stranded products, so the application of the HAND system in the technical fields of gene chips and probe melting curve analysis is limited.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] Therefore, in order to meet the clinical requirements for the simultaneous amplification and detection of multiple target nucleic acids, it is necessary to develop a new method that can simultaneously achieve efficient multiplex PCR amplification and asymmetric PCR amplification and can asymmetrically amplify multiple target nucleic acids simultaneously. [Means for Solving the Problems]

[0007] In this application, unless otherwise specified, the scientific and technical terms used in this specification have the meanings generally understood by those skilled in the art. Furthermore, all the operating steps of the nucleic acid chemistry laboratory used in this specification are common steps widely used in the corresponding field. On the other hand, for a better understanding of the present invention, the definitions and explanations of related terms are shown below.

[0008] As used in this specification, the terms "target nucleic acid sequence", "target nucleic acid" and "target sequence" refer to the target nucleic acid to be detected or its sequence. In this application, the terms "target nucleic acid sequence", "target nucleic acid" and "target sequence" have the same meaning and are used interchangeably.

[0009] As used herein, the terms "target nucleic acid specific sequence" and "target specific sequence" refer to a sequence that can selectively / specifically hybridize or anneal to a target nucleic acid under conditions that allow nucleic acid hybridization, annealing, or amplification, and include a complementary sequence to the target nucleic acid sequence. In this application, the terms "target nucleic acid specific sequence" and "target specific sequence" have the same meaning and are used interchangeably. It can be easily understood that the target nucleic acid specific sequence or the target specific sequence is specific to the target nucleic acid. In other words, the target nucleic acid specific sequence or the target specific sequence can hybridize or anneal only to a specific target nucleic acid under conditions that allow nucleic acid hybridization, annealing, or amplification, and cannot hybridize or anneal to other nucleic acid sequences. For example, in this application, the "target nucleic acid specific forward nucleotide sequence" refers to a forward nucleic acid that can selectively / specifically hybridize or anneal to a target nucleic acid under conditions that allow nucleic acid hybridization, annealing, or amplification, and includes a sequence complementary to the target nucleic acid.

[0010] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds with each other according to the principle of base pairing (Watson-Crick principle), thereby forming a duplex. In the present application, the term "complementary" includes "substantially complementary" and "fully complementary". As used herein, the term "fully complementary" means that all bases in one nucleic acid sequence can pair with the bases of another nucleic acid strand without mismatch or gap. As used herein, the term "substantially complementary" means that most of the bases in one nucleic acid sequence can pair with the bases of another nucleic acid strand, and the presence of mismatches or gaps (e.g., mismatches or gaps of one or more nucleotides) is allowed. Typically, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or fully complementary) hybridize or anneal selectively / specifically under conditions that allow nucleic acid hybridization, annealing, or amplification to form a duplex. Thus, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal under conditions that allow them to form a duplex by nucleic acid hybridization, annealing, or amplification. As used herein, the term "not fully complementary" means that the bases of a nucleic acid sequence cannot pair completely with the bases of another nucleic acid strand, and at least one mismatch or gap is present.

[0011] As used herein, the term "substitution" means that a particular nucleotide in a DNA molecule is replaced by another nucleotide. Generally, substitutions can be divided into two categories: transitions and transversions. Transitions refer to the replacement of one purine nucleotide by another purine nucleotide, or the replacement of one pyrimidine nucleotide by another pyrimidine nucleotide (e.g., replacement of A by G, G by A, C by T, T by C). Transversions refer to the replacement of a purine nucleotide by a pyrimidine nucleotide, or the replacement of a pyrimidine nucleotide by a purine nucleotide (e.g., replacement of A by T or C, G by T or C, T by A or G, C by A or G).

[0012] As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, particularly the temperature.

[0013] As used herein, the term "conditions allowing nucleic acid hybridization" has the meaning generally understood by those skilled in the art and can be determined by conventional methods. For example, two nucleic acid molecules having complementary sequences can hybridize under appropriate hybridization conditions. Such hybridization conditions may include factors such as the temperature, pH, composition, and ionic strength of the hybridization buffer, and can be determined based on the lengths and GC contents of the two complementary nucleic acid molecules. For example, when the lengths of two complementary nucleic acid molecules are relatively short and / or the GC content is relatively low, low-stringency hybridization conditions can be used. When the lengths of two complementary nucleic acid molecules are relatively long and / or the GC content is relatively high, high-stringency hybridization conditions can be used. Such hybridization conditions are known to those skilled in the art and can be found, for example, in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Therefore, the expressions "conditions allowing nucleic acid hybridization" and "conditions allowing nucleic acid annealing" also have the same meaning and are used interchangeably.

[0014] As used herein, the expression "state capable of nucleic acid amplification" has the meaning generally understood by those skilled in the art, which refers to the condition under which a nucleic acid polymerase (e.g., DNA polymerase) can use one nucleic acid strand as a template to synthesize another nucleic acid strand and form a duplex. Such conditions are known to those skilled in the art and may be related to factors such as the temperature, pH, composition, concentration, and ionic strength of the hybridization buffer, among others. Appropriate nucleic acid amplification conditions can be determined by routine methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). In the method of the present invention, the "conditions capable of nucleic acid amplification" are preferably the working conditions of a nucleic acid polymerase (e.g., DNA polymerase).

[0015] As used herein, the expression "conditions under which a nucleic acid polymerase can perform an extension reaction" has the meaning generally understood by those skilled in the art, which refers to conditions under which a nucleic acid polymerase (e.g., DNA polymerase) can use a nucleic acid strand as a template to extend another nucleic acid strand (such as a primer or a probe) to form a duplex. Such conditions are known to those skilled in the art and may be related to factors such as the temperature, pH, composition, concentration, and ionic strength of the hybridization buffer. Appropriate nucleic acid amplification conditions can be determined by routine methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). In the method of the present invention, the "conditions under which a nucleic acid polymerase can perform an extension reaction" are preferably the operating conditions of the nucleic acid polymerase (e.g., DNA polymerase). In this application, the expressions "conditions under which a nucleic acid polymerase can perform an extension reaction" and "conditions under which nucleic acid extension is possible" have the same meaning and are used interchangeably.

[0016] The operating conditions of various enzymes can be determined by those skilled in the art by routine methods and generally may include factors such as temperature, pH, composition, concentration, and ionic strength of the buffer. Alternatively, the conditions recommended by the enzyme manufacturer can also be used.

[0017] As used herein, the term "nucleic acid denaturation" has the meaning generally understood by those skilled in the art and refers to the process by which a double-stranded nucleic acid molecule dissociates into single strands. The expression "conditions capable of nucleic acid denaturation" refers to the conditions under which a double-stranded nucleic acid molecule dissociates into single strands. Such conditions can be determined by those skilled in the art according to conventional methods (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). For example, nucleic acids can be denatured by conventional techniques such as heating, alkaline treatment, urea treatment, enzymatic methods (such as methods using helicase), etc. In the present application, the nucleic acid is preferably denatured under heating. For example, the nucleic acid can be denatured by heating it to 80°C to 105°C.

[0018] As used herein, the term "PCR reaction" has the meaning generally understood by those skilled in the art and refers to a reaction (polymerase chain reaction) for amplifying a target nucleic acid using a nucleic acid polymerase and primers. As used herein, the term "multiplex amplification" refers to the amplification of multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to the fact that in the amplification product obtained by amplifying a target nucleic acid, the amounts of two complementary nucleic acid strands are different, and the amount of one nucleic acid strand is more than that of the other nucleic acid strand.

[0019] As used herein and as generally understood by those skilled in the art, the terms "forward" and "reverse" are used for convenience only when describing and distinguishing the two primers of a primer pair, and they are relative and have no special meaning.

[0020] As used herein, the term "fluorescent probe" refers to an oligonucleotide having a fluorescent group and capable of generating a fluorescent signal.

[0021] As used herein, the term "melting curve analysis" has the meaning generally understood by those skilled in the art and refers to a method of analyzing the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule, which is commonly used to assess the dissociation characteristics of a double-stranded nucleic acid molecule during heating. Methods for performing melting curve analysis are known to those skilled in the art (see, for example, The Journal of Molecular Diagnostics 2009, 11(2):93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

[0022] In certain preferred embodiments of the present application, melting curve analysis can be performed using detection probes labeled with a reporter group and a quencher group. Briefly, at ambient temperature, the detection probe can form a duplex with its complementary sequence via base pairing. In this case, the reporter group (such as a fluorophore) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescence signal) from the reporter group. At this point, the strongest signal (such as a fluorescence signal) can be detected. As the temperature rises, the two strands of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe exists in a single-stranded free coil state. In this case, the reporter group (such as a fluorophore) and the quencher group on the dissociated detection probe are close to each other, whereby the signal (such as a fluorescence signal) emitted by the reporter group (such as a fluorophore) is absorbed by the quencher group. Therefore, as the temperature rises, the detected signal (such as a fluorescence signal) gradually weakens. When the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all signals (such as fluorescence signals) emitted by the reporter group (such as a fluorophore) on the detection probe are absorbed by the quencher group. Therefore, the signal (such as a fluorescence signal) emitted by the reporter group (such as a fluorophore) is essentially undetectable. Therefore, by detecting the signal (such as a fluorescence signal) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation processes between the detection probe and its complementary sequence can be observed, and a curve is formed as the signal intensity changes with temperature. Furthermore, by performing a derivative analysis of the obtained curve, a curve with the signal intensity change rate as the vertical axis and the temperature as the horizontal axis (i.e., the melting curve of the duplex) can be obtained. The peak of the melting curve is the melting peak, and the corresponding temperature is the melting point (T m) It is. Generally, the higher the degree of match between the detection probe and the complementary sequence (for example, the fewer the mismatched bases and the more the paired bases), the higher the T of the duplex m becomes. Therefore, by detecting the T of the duplex m , the presence and identity of the complementary sequence to the detection probe within the duplex can be determined. As used herein, the terms "melting peak," "melting point," and "T m " have the same meaning and are used interchangeably.

[0023] Amplification method In one aspect, the present invention is a method for amplifying one or more target nucleic acids in a sample, comprising (1) providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair for each target nucleic acid to be amplified, wherein the universal primer contains a first universal sequence, the target-specific primer pair can amplify the target nucleic acid and includes a forward primer and a reverse primer, the forward primer contains a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid, the forward nucleotide sequence is located at the 3'-end of the second universal sequence, the reverse primer contains a first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, the reverse nucleotide sequence is located at the 3'-end of the first universal sequence, under conditions allowing nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence, there is a difference between the second universal sequence and the first universal sequence, the difference includes that one or more nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted, and the first universal sequence is not completely complementary to the complementary sequence of the forward primer (2) Under conditions where nucleic acid amplification is possible, amplifying the target nucleic acid in the sample via a PCR reaction using a universal primer and a target-specific primer pair; A method comprising the same is provided.

[0024] In the method of the present invention, the forward primer and the reverse primer each contain a forward nucleotide sequence and a reverse nucleotide sequence specific to the target nucleic acid. Thus, during the PCR reaction, the target-specific primer pair (forward primer and reverse primer) anneals to the target nucleic acid to initiate PCR amplification, resulting in an initial amplification product containing two nucleic acid strands (nucleic acid strand A and nucleic acid strand B) complementary to the forward primer and the reverse primer, respectively.

[0025] Furthermore, since both the reverse primer and the universal primer contain a first universal sequence, nucleic acid strand B complementary to the reverse primer can also be made complementary to the universal primer. Therefore, during the PCR reaction, the universal primer anneals to nucleic acid strand B and can usually initiate PCR amplification (i.e., usually synthesize the complementary strand of nucleic acid strand B). At the same time, since the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence under conditions where nucleic acid hybridization or annealing is possible, during the PCR reaction, the universal primer (containing the first universal sequence) can also anneal to nucleic acid strand A complementary to the forward primer (containing the second universal sequence). However, since the second universal sequence is different from the first universal sequence (in this case, one or more nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted), the universal primer (especially their 3'-ends) is not completely complementary to nucleic acid strand A, resulting in inhibition of PCR amplification of nucleic acid strand A by the universal primer (i.e., synthesis of the complementary strand of nucleic acid strand A is inhibited).

[0026] Therefore, as the PCR reaction progresses, the universal primers anneal to nucleic acid strand A and nucleic acid strand B of the initial amplification products respectively, and further initiate PCR amplification. While the synthesis of the complementary strand of nucleic acid strand B proceeds normally, the synthesis of the complementary strand of nucleic acid strand A is inhibited. Therefore, as the PCR amplification progresses, the synthesis efficiency of the complementary strand of nucleic acid strand A (nucleic acid strand B) becomes significantly lower than that of the complementary strand of nucleic acid strand B (nucleic acid strand A). As a result, a large amount of the complementary strand of nucleic acid strand B (nucleic acid strand A) is synthesized and amplified, while the synthesis and amplification of the complementary strand of nucleic acid strand A (nucleic acid strand B) are inhibited, and a large amount of single-stranded products (nucleic acid strand A containing a sequence complementary to the forward primer / second universal primer sequence and a sequence of the reverse primer / universal primer) are generated, thereby enabling the asymmetric amplification of the target nucleic acid. Therefore, in a certain specific preferred embodiment, the method of the present invention enables the asymmetric amplification of one or more target nucleic acids in a sample.

[0027] Furthermore, since the first universal sequence in the reverse primer can hybridize or anneal to the complementary sequence of the second universal sequence in the forward primer under conditions allowing nucleic acid hybridization or annealing, during the PCR reaction, the primer dimers formed by the non-specific amplification of the forward primer and the reverse primer, after denaturation, generate single-stranded nucleic acids with complementary 5'-ends and 3'-ends that can anneal to each other. The single-stranded nucleic acids are prone to self-annealing during the annealing stage to form a stable panhandle structure, preventing the universal primer from annealing and extending the single-stranded nucleic acid, thereby inhibiting further amplification of the primer dimer. Therefore, in the method of the present invention, the non-specific amplification of the primer dimer can be effectively suppressed. Therefore, in the method of the present invention, for example, the universal primer can be used in combination with one or more target-specific primer pairs to achieve the multiplex amplification of one or more target nucleic acids.

[0028] Thus, in certain preferred embodiments, the method of the present invention can simultaneously amplify from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target nucleic acids, for example at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target nucleic acids. In certain preferred embodiments, the method of the present invention can simultaneously asymmetrically amplify from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target nucleic acids. In such embodiments, therefore, for each target nucleic acid to be amplified, one target-specific primer pair is provided in step (1). Thus, in such embodiments, in step (1), from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target-specific primer pairs, for example at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target-specific primer pairs are provided.

[0029] It can be readily understood that different forward and reverse primers can be used for different target nucleic acids. However, when there is sequence similarity between different target nucleic acids, different target-specific primer pairs may have the same forward or reverse primer.

[0030] In order to facilitate multiple asymmetric amplifications and effectively suppress non-specific amplifications of primer dimers, in some preferred embodiments, the universal primer has an operating concentration higher than the operating concentrations of the forward primer and the reverse primer. In certain preferred embodiments, the universal primer has an operating concentration at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 18-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold or higher than the operating concentrations of the forward primer and the reverse primer. In certain preferred embodiments, the universal primer has an operating concentration 1-fold to 5-fold, 5-fold to 10-fold, 10-fold to 15-fold, 15-fold to 20-fold, 20-fold to 50-fold or higher than the operating concentrations of the forward primer and the reverse primer.

[0031] In the method of the present invention, the operating concentrations of the forward primer and the reverse primer may be the same or different. In certain preferred embodiments, the operating concentrations of the forward primer and the reverse primer are the same. In certain preferred embodiments, the operating concentrations of the forward primer and the reverse primer are different. In certain preferred embodiments, the operating concentration of the forward primer is lower than the operating concentration of the reverse primer.

[0032] In certain preferred embodiments, the universal primer consists of a first universal sequence. In certain preferred embodiments, the universal primer further comprises an additional sequence located at the 5' end of the first universal sequence. In certain preferred embodiments, the additional sequence comprises one or more nucleotides, such as 1 to 5, 5 to 10, 10 to 15, 15 to 20, or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In the present application, since the universal primer is used for PCR amplification, preferably, the first universal sequence is located at the 3' portion of the universal primer or constitutes the 3' portion of the universal primer.

[0033] In the embodiments of the present application, the universal primer may have any length as long as it can perform a PCR reaction. For example, the universal primer may have a length of 5 nt to 50 nt, such as 5 nt to 15 nt, 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, or 40 nt to 50 nt.

[0034] In certain embodiments of the present application, a universal primer (or any of its components) may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleosides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the universal primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the universal primer (or any of its components) comprises modified nucleotides, e.g., modified deoxyribonucleotides or ribonucleotides such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the universal primer (or any of its components) comprises unnatural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA). In certain embodiments of the present application, in step (1), at least one target-specific primer pair, e.g., at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target-specific primer pairs are provided. In certain preferred embodiments, in step (1), 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50, or more target-specific primer pairs are provided. In certain preferred embodiments, the above method can simultaneously amplify 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50, or more target nucleic acids. In certain preferred embodiments, the above method can simultaneously asymmetrically amplify 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50, or more target nucleic acids.

[0035] For example, in a certain specific preferred embodiment, the method of the present invention can be used to amplify two target nucleic acids in a sample. In step (1), a universal primer, a first target-specific primer pair, and a second target-specific primer pair are provided. The universal primer is as defined above. The first target-specific primer pair includes a first forward primer and a first reverse primer that can amplify the first target nucleic acid. The second target-specific primer pair includes a second forward primer and a second reverse primer that can amplify the second target nucleic acid. The first forward primer, the first reverse primer, the second forward primer, and the second reverse primer are as defined above. Similarly, in a certain specific preferred embodiment, the method of the present invention can be used to amplify three or more target nucleic acids in a sample. In step (1), a universal primer and three or more target-specific primer pairs that can amplify three or more target nucleic acids are provided.

[0036] In a certain specific preferred embodiment, in the forward primer, the forward nucleotide sequence is directly linked to the 3'-end of the second universal sequence. In a certain specific preferred embodiment, in the forward primer, the forward nucleotide sequence is linked to the 3'-end of the second universal sequence by a nucleotide linker. In a certain specific preferred embodiment, the forward primer contains or consists of, from 5' to 3', the second universal sequence and the forward nucleotide sequence. In a certain specific preferred embodiment, the forward primer contains or consists of, from 5' to 3', the second universal sequence, a nucleotide linker, and the forward nucleotide sequence. In a certain specific preferred embodiment, the nucleotide linker contains one or more nucleotides, for example, 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0037] In certain preferred embodiments, the forward primer further comprises an additional sequence located at the 5' end of the second universal sequence. Thus, in certain preferred embodiments, the forward primer comprises, or consists of, from 5' to 3', the additional sequence, the second universal sequence, and the forward nucleotide sequence. In certain preferred embodiments, the forward primer comprises, or consists of, from 5' to 3', the additional sequence, the second universal sequence, a nucleotide linker, and the forward nucleotide sequence. In certain preferred embodiments, the additional sequence comprises one or more nucleotides, such as from 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0038] In the present application, since the forward primer is used for PCR amplification of the target nucleic acid, preferably, the forward nucleotide sequence is located at the 3' portion of the forward primer or constitutes the 3' portion of the forward primer.

[0039] In the embodiments of the present application, the forward nucleotide sequence is not limited by its length as long as it can specifically hybridize with the target nucleic acid sequence and amplify the target nucleic acid. For example, the forward nucleotide sequence can have a length of 10 nt to 100 nt, such as 10 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt.

[0040] In the embodiments of the present application, the forward primer is not limited by its length as long as it satisfies the conditions defined above. For example, the forward primer can have a length of 15 nt to 150 nt, such as 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, 100 nt to 110 nt, 110 nt to 120 nt, 120 nt to 130 nt, 130 nt to 140 nt, 140 nt to 150 nt.

[0041] In certain specific embodiments of the present application, the forward primer (or any of its components) can comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the forward primer (or any of its components) comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the forward primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides like 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the forward primer (or any of its components) comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0042] In certain preferred embodiments, in the reverse primer, the reverse nucleotide sequence is directly linked to the 3'-end of the first universal sequence. In certain preferred embodiments, in the reverse primer, the reverse nucleotide sequence is linked to the 3'-end of the first universal sequence by a nucleotide linker. In certain preferred embodiments, the reverse primer comprises or consists of, from 5' to 3', the first universal sequence and the reverse nucleotide sequence. In certain preferred embodiments, the reverse primer comprises or consists of, from 5' to 3', the first universal sequence, a nucleotide linker, and the reverse nucleotide sequence. In certain preferred embodiments, the nucleotide linker comprises one or more nucleotides, for example, 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0043] In certain preferred embodiments, the reverse primer further comprises an additional sequence located at the 5'-end of the first universal sequence. Thus, in certain preferred embodiments, the reverse primer comprises or consists of, from 5' to 3', the additional sequence, the first universal sequence, and the reverse nucleotide sequence. In certain preferred embodiments, the reverse primer comprises or consists of, from 5' to 3', the additional sequence, the first universal sequence, a nucleotide linker, and the reverse nucleotide sequence. In certain preferred embodiments, the additional sequence comprises one or more nucleotides, for example, 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0044] In the present application, since the reverse primer is used for PCR amplification of the target nucleic acid, preferably, the reverse nucleotide sequence is located at the 3'-portion of the reverse primer or constitutes the 3'-portion of the reverse primer.

[0045] In an embodiment of the present application, the reverse nucleotide sequence is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence and amplify the target nucleic acid. For example, the reverse nucleotide sequence may have a length of 10 nt to 100 nt, such as 10 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt.

[0046] In an embodiment of the present application, the reverse primer is not limited by its length as long as it satisfies the conditions defined above. For example, the reverse primer may have a length of 15 nt to 150 nt, such as 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, 100 nt to 110 nt, 110 nt to 120 nt, 120 nt to 130 nt, 130 nt to 140 nt, 140 nt to 150 nt.

[0047] In certain embodiments of the present application, a reverse primer (or any component thereof) can comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides, or any combination thereof. In certain preferred embodiments, the reverse primer (or any component thereof) comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the reverse primer (or any component thereof) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, for example, 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the reverse primer (or any component thereof) comprises unnatural nucleotides, such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0048] In an embodiment of the present application, the second universal sequence in the forward primer is different from the first universal sequence in the universal primer, and the difference includes that one or more nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that one or more nucleotides (for example, 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) located at the 3'-end of the first universal sequence are independently deleted or substituted, or there is a difference there. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that 1, 2, 3, 4, or 5 nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted, or there is a difference there. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that the last nucleotide at the 3'-end of the first universal sequence is deleted or substituted, or there is a difference there. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that the last two nucleotides located at the 3'-end of the first universal sequence are deleted or substituted, or there is a difference there. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that the last nucleotide at the 3'-end of the first universal sequence is deleted and the penultimate nucleoside is substituted, or there is a difference there. In a certain preferred embodiment, the difference between the second universal sequence and the first universal sequence includes that the last nucleotide at the 3'-end of the first universal sequence is substituted and the penultimate nucleoside is deleted, or there is a difference there.In certain preferred embodiments, the difference between the second universal array and the first universal array includes that the last three, last four, or last five nucleotides located at the 3' end of the first universal array are each independently deleted or substituted, or there is a difference there. In the present application, the substitution can be a transition or a transversion. In certain preferred embodiments, the substitution is a transition. In certain preferred embodiments, the substitution is a transversion.

[0049] In the embodiments of the present application, the first universal array is not completely complementary to the complementary sequence of the forward primer. In certain preferred embodiments, at least one nucleotide located at the 3' end of the first universal array, for example, 1 to 5, 5 to 10, 10 to 15, 15 to 20, or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, should not be complementary to the complementary sequence of the forward primer. Therefore, during the PCR reaction, even if the first universal array / universal primer can anneal to the nucleic acid strand (nucleic acid strand A) complementary to the forward primer, the extension synthesis of the complementary strand of the nucleic acid strand (nucleic acid strand A) is still inhibited.

[0050] In the method of the present invention, the sample can be any sample containing nucleic acids. For example, in certain preferred embodiments, the sample contains DNA (e.g., genomic DNA or cDNA), or is DNA. In certain preferred embodiments, the sample contains RNA (e.g., mRNA), or is RNA. In certain preferred embodiments, the sample contains a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA), or is a mixture of nucleic acids.

[0051] In the method of the present invention, the target nucleic acid to be amplified is not restricted by the composition or length of its sequence. For example, the target nucleic acid can be DNA (e.g., genomic DNA or cDNA) or an RNA molecule (e.g., mRNA). Furthermore, the target nucleic acid to be amplified may be single-stranded or double-stranded.

[0052] When the sample or the target nucleic acid is mRNA, preferably, before performing the method of the present invention, a reverse transcription reaction is carried out to obtain cDNA complementary to the mRNA. A detailed description of the reverse transcription reaction can be found, for example, in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

[0053] In the method of the present invention, the sample or the target nucleic acid can be obtained from any source including, but not limited to, prokaryotes, eukaryotes (e.g., animals including protozoa, parasites, fungi, yeast, plants, mammals, and humans), or viruses (e.g., herpes virus, HIV, influenza virus, Epstein - Barr virus, hepatitis virus, poliovirus, etc.), or viroids. The sample or the target nucleic acid can also be any form of nucleic acid such as genomic nucleic acid, artificially isolated or fragmented nucleic acid, synthetic nucleic acid, etc.

[0054] In the method of the present invention, any nucleic acid polymerase (especially template-dependent nucleic acid polymerase) can be used to perform a PCR reaction. In certain preferred embodiments, the nucleic acid polymerase is a DNA polymerase. In certain preferred embodiments, the nucleic acid polymerase is a heat-resistant DNA polymerase. Heat-resistant DNA polymerases include, but are not limited to, Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococto gorgonarius, Thermotoga maritima, Thermotoga maritimaIt can be obtained from various bacterial species such as Thermosipho africanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus, and Aquifex aeolicus. In particular, the DNA polymerase is preferably Taq polymerase.

[0055] In certain preferred embodiments, the target nucleic acid is amplified in a three-step process. In such embodiments, each round of nucleic acid amplification requires three steps: nucleic acid denaturation at a first temperature, nucleic acid annealing at a second temperature, and nucleic acid extension at a third temperature. In certain preferred embodiments, the target nucleic acid is amplified in a two-step process. In such embodiments, each round of nucleic acid amplification requires two steps: nucleic acid denaturation at a first temperature and nucleic acid annealing and extension at a second temperature. The temperatures suitable for nucleic acid denaturation, nucleic acid annealing, and nucleic acid extension can be readily determined by those skilled in the art in routine ways (see, for example, Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)).

[0056] In certain preferred embodiments, steps (1) and (2) of the method of the present invention are the following steps (a) to (f): (a) Providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair for each target nucleic acid to be amplified, wherein the universal primer and the target-specific primer pair are as defined above; (b) Mixing the sample with a universal primer and a target-specific primer pair and a nucleic acid polymerase (e.g., a template-dependent nucleic acid polymerase; DNA polymerase, etc., particularly a thermostable DNA polymerase); (c) Incubating the product of the previous step under conditions capable of nucleic acid denaturation; (d) Incubating the product of the previous step under conditions capable of nucleic acid annealing or hybridization; (e) Incubating the product of the previous step under conditions capable of nucleic acid extension; (f) Optionally, repeating steps (c) to (e) one or more times; It can be carried out by a protocol including the above steps.

[0057] In such an embodiment, in step (c), all nucleic acid molecules in the sample are dissociated into single-stranded states, and then, in step (d), complementary nucleic acid molecules (for example, a forward primer and a target nucleic acid or an extension product of a reverse primer, a reverse primer and a target nucleic acid or an extension product of a forward primer, an amplification product containing a universal primer and a complementary sequence of a first universal sequence, an amplification product containing a universal primer and a complementary sequence of a second universal sequence) anneal or hybridize together to form a duplex. Then, in step (e), a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) extends the forward primer / reverse primer and the universal primer that hybridize to the complementary sequence. In this process, as described above, the nucleic acid polymerase can use nucleic acid strand B as a template, usually extends the universal primer, and can synthesize a complementary strand of nucleic acid strand B. However, since the universal primer (particularly its 3'-end) cannot be completely complementary to nucleic acid strand A, the extension of the universal primer by the nucleic acid polymerase using nucleic acid strand A as a template is inhibited (that is, the synthesis of the complementary strand of nucleic acid strand A is inhibited). Therefore, through the cycles of steps (c) to (e), amplification (asymmetric amplification) of the target nucleic acid sequence is achieved, whereby steps (1) and (2) of the method of the present invention can be completed.

[0058] The incubation time and temperature in step (c) can be determined by those skilled in the art according to the conventional method. In a certain specific preferred embodiment, in step (c), the product of step (b) is incubated at a temperature of 80°C to 105°C (for example, 80°C to 85°C, 85°C to 90°C, 90°C to 95°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, or 105°C), thereby enabling the denaturation of nucleic acids. In a certain specific preferred embodiment, in step (c), the product of step (b) is incubated for 10 seconds to 5 minutes, for example, 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, or 2 minutes to 5 minutes.

[0059] The incubation time and temperature of step (d) can be determined by those skilled in the art according to conventional methods. In certain preferred embodiments, in step (d), the product of step (c) is incubated at a temperature of 35°C to 70°C (for example, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 60°C to 65°C, or 65°C to 70°C), thereby enabling nucleic acid annealing or hybridization. In certain preferred embodiments, in step (d), the product of step (c) is incubated for 10 seconds to 5 minutes, for example, 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, or 2 minutes to 5 minutes.

[0060] The incubation time and temperature of step (e) can be determined by those skilled in the art according to conventional methods. In certain preferred embodiments, in step (e), the product of step (d) is incubated at a temperature of 35°C to 85°C (for example, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 60°C to 65°C, 65°C to 70°C, 70°C to 75°C, 75°C to 80°C, 80°C to 85°C), thereby enabling nucleic acid elongation. In certain preferred embodiments, in step (e), the product of step (d) is incubated for 10 seconds to 30 minutes, for example, 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, 2 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, or 20 minutes to 30 minutes.

[0061] In certain embodiments, steps (d) and (e) may be performed at different temperatures, that is, nucleic acid annealing and elongation are performed at different temperatures. In certain embodiments, steps (d) and (e) may be performed at the same temperature, that is, nucleic acid annealing and elongation are performed at the same temperature. In this case, steps (d) and (e) can be combined as one step.

[0062] In the method of the present invention, steps (c) to (e) may be repeated at least once, for example, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. However, when steps (c) to (e) are repeated more than once, it can be easily understood that the conditions used in steps (c) to (e) of each cycle do not have to be the same. For example, using certain conditions, steps (c) to (e) of the previous part of the cycle (e.g., the first 5 cycles, the first 10 cycles, the first 20 cycles) can be carried out, and then steps (c) to (e) of the remaining cycles can be carried out using different conditions.

[0063] The method of the present invention enables multiplex asymmetric amplification of target nucleic acids and enables the production of a large amount of single-stranded nucleic acid products. Therefore, the method of the present invention is particularly advantageous in certain situations. For example, the amplification products of the method of the present invention can be used for sequencing, gene chip detection, or melting curve analysis. Therefore, in some preferred embodiments, the method of the present invention further includes the step of (3) sequencing the product of step (2). In certain specific preferred embodiments, the method of the present invention further includes the step of (3) detecting the product of step (2) using a gene chip. In certain specific preferred embodiments, the method of the present invention further includes the step of (3) performing melting curve analysis on the product of step (2).

[0064] Detection method In one aspect, the present application provides a method for detecting one or more target nucleic acids in a sample, including the steps of (i) amplifying one or more target nucleic acids in the sample using the method of the present invention, and (ii) performing melting curve analysis on the product of step (i).

[0065] In certain specific preferred embodiments, the method of the present invention (1) providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair and a detection probe for each target nucleic acid to be amplified, The universal primer contains a first universal sequence, The target-specific primer pair can amplify the target nucleic acid, and includes a forward primer and a reverse primer. The forward primer contains a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid. The forward nucleotide sequence is located at the 3' end of the second universal sequence. The reverse primer contains a first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid. The reverse nucleotide sequence is located at the 3' end of the first universal sequence. Under conditions allowing nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence. There is a difference between the second universal sequence and the first universal sequence, and the difference includes that one or more nucleotides located at the 3' end of the first universal sequence are independently deleted or substituted. The first universal sequence shall not be completely complementary to the complementary sequence of the forward primer. The detection probe contains a probe nucleotide sequence specific to the target nucleic acid and is labeled with a reporter group and a quencher group. The reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group. The signal emitted when the detection probe hybridizes to its complementary sequence is different from the signal emitted when it is not hybridized to its complementary sequence. (2) Under conditions allowing nucleic acid amplification, using the universal primer and the target-specific primer pair, amplifying the target nucleic acid in the sample through a PCR reaction. (3) Using the detection probe to perform melting curve analysis on the product of step (2), and determining whether the target nucleic acid is present in the sample according to the result of the melting curve analysis. including.

[0066] In a particular embodiment of the present invention, the sample, the target nucleic acid, the universal primer, and / or the target-specific primer pair are as defined above.

[0067] In a particular embodiment of the present invention, in step (2), the sample is mixed with the universal primer and the target-specific primer pair and a nucleic acid polymerase, and a PCR reaction is performed. Then, after the PCR reaction is completed, a detection probe is added to the product of step (2), and melting curve analysis is performed.

[0068] In a particular embodiment of the present invention, in step (2), the sample is mixed with the universal primer, the target-specific primer pair, and the detection probe and a nucleic acid polymerase, and a PCR reaction is performed. Then, after the PCR reaction is completed, melting curve analysis is performed.

[0069] In a particular embodiment of the present invention, the detection probe may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, unnatural nucleotides (e.g., peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof. In a particular preferred embodiment, the detection probe comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In a particular preferred embodiment, the detection probe comprises modified nucleotides, e.g., modified deoxyribonucleotides or ribonucleotides such as 5-methylcytosine or 5-hydroxymethylcytosine. In a particular preferred embodiment, the detection probe comprises unnatural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, or locked nucleic acid (LNA).

[0070] In the method of the present invention, the detection probe is not limited by its length. For example, the detection probe has a length of 15 nt to 1000 nt, such as 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, 100 nt to 200 nt, 200 nt to 300 nt, 300 nt to 400 nt, 400 nt to 500 nt, 500 nt to 600 nt, 600 nt to 700 nt, 700 nt to 800 nt, 800 nt to 900 nt, 900 nt to 1000 nt.

[0071] In certain preferred embodiments, the detection probe has a 3'-OH terminus. In certain preferred embodiments, the 3' terminus of the detection probe is blocked to inhibit its extension. The 3' terminus of a nucleic acid (such as a detection probe) can be blocked in various ways. For example, the 3' terminus of the detection probe can be blocked by modifying the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, the 3' terminus of the detection probe can be blocked by adding a chemical moiety (such as biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, the 3' terminus of the detection probe can be blocked by removing the 3'-OH of the last nucleotide of the detection probe or by replacing the last nucleotide with a dideoxynucleotide.

[0072] As described above, the detection probe is labeled with a reporter group and a quencher group, the reporter group can emit a signal, the quencher group can absorb or quench the signal emitted by the reporter group, and the signal emitted when the detection probe hybridizes to its complementary sequence is different from the signal emitted when it is not hybridized to its complementary sequence.

[0073] In certain preferred embodiments, the detection probe is a self-quenching probe. In such embodiments, when the detection probe is not hybridized to another sequence, the quencher group is positioned such that it can absorb or quench the signal of the reporter group (e.g., the quencher group is positioned adjacent to the reporter group), thereby absorbing or quenching the signal emitted from the reporter group. In this case, the detection probe does not emit a signal. Further, when the detection probe hybridizes to its complementary sequence, the quencher group is in a position where it cannot absorb or quench the signal of the reporter group (e.g., the quencher group is far from the reporter group), and thus cannot absorb or quench the signal emitted from the reporter group. In this case, the detection probe emits a signal.

[0074] The design of such self-quenching detection probes is within the capabilities of those skilled in the art. For example, the reporter group can be labeled at the 5' end of the detection probe and the quencher group can be labeled at the 3' end, or the reporter group can be labeled at the 3' end of the detection probe and the quencher group can be labeled at the 5' end. Thus, when the detection probe exists alone, the reporter group and the quencher group interact closely with each other, so the signal emitted by the reporter group is absorbed by the quencher group and the detection probe does not emit a signal. Also, when the detection probe hybridizes to its complementary sequence, the reporter group and the quencher group are separated from each other, so the signal emitted by the reporter group cannot be absorbed by the quencher group and the detection probe emits a signal.

[0075] However, it should be understood that the reporter group and the quencher group do not have to be labeled at both ends of the detection probe. The reporter group and / or the quencher group can also be internally labeled within the detection probe as long as the detection probe emits a signal different from the signal emitted when not hybridized to its complementary sequence when hybridized to its complementary sequence. For example, the reporter group can be labeled upstream (or downstream) of the detection probe, and the quencher group can be labeled downstream (or upstream) of the detection probe, and the two are separated by a sufficient distance (e.g., 10 nt - 20 nt, 20 nt - 30 nt, 30 nt - 40 nt, 40 nt - 50 nt, 50 nt - 60 nt, 60 nt - 70 nt, 70 nt - 80 nt, or a longer distance). Therefore, when the detection probe exists alone, due to the formation of the free coil of the probe molecule or the formation of the secondary structure of the probe (e.g., hairpin structure), the reporter group and the quencher group approach and interact with each other, so the signal emitted by the reporter group is absorbed by the quencher group. As a result, the detection probe does not emit a signal. When the detection probe hybridizes to its complementary sequence, the reporter group and the quencher group are separated from each other by a sufficient distance so that the quencher group cannot absorb the signal emitted by the reporter group, whereby the detection probe emits a signal. In certain preferred embodiments, the reporter group and the quencher group are separated by a distance of 10 nt - 80 nt or more, e.g., 10 nt - 20 nt, 20 nt - 30 nt, 30 nt - 40 nt, 40 nt - 50 nt, 50 nt - 60 nt, 60 nt - 70 nt, 70 nt - 80 nt. In certain preferred embodiments, the reporter group and the quencher group are separated by a distance of 80 nt or less, 70 nt or less, 60 nt or less, 50 nt or less, 40 nt or less, 30 nt or less, or 20 nt or less. In certain preferred embodiments, the reporter group and the quencher group are separated by a distance of at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt.

[0076] Accordingly, as long as the detection probe emits a signal different from the signal emitted when not hybridized to the complementary sequence when hybridized to the complementary sequence, the reporter group and the quencher group can be labeled at any suitable position on the detection probe. However, in certain preferred embodiments, at least one of the reporter group and the quencher group is located at the end of the detection probe (e.g., the 5' or 3' end). In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe or 1 nt to 10 nt from the 5' end, and the reporter group and the quencher group are separated by an appropriate distance such that the quencher group can absorb or quench the signal emitted by the reporter group before the detection probe hybridizes to its complementary sequence. In certain preferred embodiments, one of the reporter group and the quencher group is located at the 3' end of the detection probe or 1 nt to 10 nt from the 3' end, and the reporter group and the quencher group are separated by an appropriate distance such that the quencher group can absorb or quench the signal emitted by the reporter group before the detection probe hybridizes to its complementary sequence. In certain preferred embodiments, the reporter group and the quencher group may be separated by the distance defined above (e.g., a distance of 10 nt to 80 nt or more). In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe and the other is located at the 3' end.

[0077] In the method of the present invention, the reporter group and the quencher group can be any suitable group or molecule known in the art, and specific examples thereof include, but are not limited to, Cy2 (trademark) (506), YO-PRO (trademark)-l (509), YOYO (trademark)-l (509), calcein (517), FITC (518), FluorX (trademark) (519), Alexa (trademark) (520), rhodamine 110 (520), Oregon Green (trademark) 500 (522), Oregon Green (trademark) 488 (524), RiboGreen (trademark) (525), Rhodamine Green (trademark) (527), rhodamine 123 (529), Magnesium Green (trademark) (531), Calcium Green (trademark) (533), TO-PRO (trademark)-l (533), TOTOl (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568), BODIPY564 / 570 (570), Cy3 (trademark) (570), Alexa (trademark) 546 (570), TRITC (572), Magnesium Orange (trademark) (575), phycoerythrin R&B (575), rhodamine phalloidin (575), Calcium Orange (trademark) (576), pyronin Y (580), rhodamine B (580), TAMRA (582), Rhodamine Red (trademark) (590), Cy3.5 (trademark) (596), ROX (608), Calcium Crimson (trademark) (615), Alexa (trademark) 594 (615), Texas Red (615), Nile Red (628), YO-PRO (trademark)-3 (631), YOYO (trademark)-3 (631), R-phycocyanin (642), C-phycocyanin (648), TO-PRO (trademark)-3 (660), T0T03 (660), DiD DilC(5) (665), Cy5 (trademark) (670), thiazicarbocyanine (671), Cy5.5(694), HEX(556), TET(536), Biosearch Blue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL Fluor Red 610(610), CAL Fluor Red 635(637), FAM(520), fluorescein(520), fluorescein-C3(520), Pulsar 650(566), Quasar 570(667), Quasar 670(705), and Quasar 705(610). The numbers in parentheses indicate the maximum emission wavelength in nm.

[0078] Furthermore, various suitable combinations of reporter groups and quencher groups are known in the art, for example, see Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Oiganic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Peigamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, RP, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patent No. 3,996,345, and U.S. Patent No. 4,351,760.

[0079] In certain preferred embodiments, the reporter group is a fluorescent group. In such embodiments, the signal emitted by the reporter group is fluorescence, and the quencher group is a molecule or group that can absorb / quench fluorescence (e.g., another fluorescent molecule that can absorb fluorescence, or a quencher that can quench fluorescence). In certain preferred embodiments, examples of fluorophores include, but are not limited to, various fluorescent molecules such as ALEX-350, FAM, VIC, TET, CAL Fluor™ Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705, etc. In certain preferred embodiments, examples of quencher groups include, but are not limited to, various quenchers such as DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA, etc.

[0080] In the method of the present invention, the detection probe can also be modified, for example, to confer resistance to nuclease activity (e.g., 5' nuclease activity, e.g., 5'→3' exonuclease activity). For example, a modification resistant to nuclease activity can be introduced into the backbone of the detection probe, and examples thereof include phosphorothioate ester bond, alkyl phosphotriester bond, aryl phosphotriester bond, alkyl phosphonate ester bond, aryl phosphonate ester bond, hydrogenated phosphate ester bond, alkyl phosphoramidate ester bond, aryl phosphoramidate ester bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification.

[0081] In the method of the present invention, the detection probe may be linear or may have a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. The hairpin structure may be natural or can be introduced artificially. Further, the detection probe having a hairpin structure can be constructed using conventional methods in the art. For example, the detection probe can form a hairpin structure by adding two complementary oligonucleotide sequences to both ends (5'-end and 3'-end) of the detection probe. In such an embodiment, the two complementary oligonucleotide sequences constitute the arms (stems) of the hairpin structure. The arms of the hairpin structure can be of any desired length. For example, the arms can have a length of 2 nt to 15 nt, such as 3 nt to 7 nt, 4 nt to 9 nt, 5 nt to 10 nt, 6 nt to 12 nt.

[0082] In certain embodiments, the product of step (2) can be gradually heated, and the signal emitted by the reporter group on the detection probe can be monitored in real time, thereby obtaining a curve of the signal intensity of the product of step (2) that varies with temperature changes. For example, the product of step (2) can be gradually heated from a temperature of 45°C or lower (e.g., 45°C or lower, 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower) to a temperature of 75°C or higher (e.g., at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C), and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve of the signal intensity of the reporter group that varies in response to temperature changes. The heating rate can be determined conventionally by those skilled in the art. For example, the heating rate can be as follows: the temperature is increased by 0.01°C to 1°C (e.g., 0.01°C to 0.05°C, 0.05°C to 0.1°C, 0.1°C to 0.5°C, 0.5°C to 1°C, 0.04°C to 4°C, e.g., 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C or 1.0°C) for each step, and each step is maintained for 0.5 seconds to 15 seconds (e.g., 0.5 seconds to 1 second, 1 second to 2 seconds, 2 seconds to 3 seconds, 3 seconds to 4 seconds, 4 seconds to 5 seconds, 5 seconds to 10 seconds, 10 seconds to 15 seconds), or the temperature is increased by 0.01°C to 1°C (e.g., 0.01°C to 0.05°C, 0.05°C to 0.1°C, 0.1°C to 0.5°C, 0.5°C to 1°C, 0.04°C to 0.4°C, e.g., 0.01°C, 0.02°C, 0.03°C, 0.04°C, 0.05°C, 0.06°C, 0.07°C, 0.08°C, 0.09°C, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, or 1.0°C) per second.

[0083] In certain specific embodiments, the product of step (2) can be gradually cooled, and the signal emitted by the reporter group of the detection probe can be monitored in real time, thereby obtaining a curve of the signal intensity of the product of step (2) that varies with temperature changes. For example, the product of step (2) can be gradually cooled from a temperature of 75 °C or higher (e.g., at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C) to a temperature of 45 °C or lower (e.g., 45 °C or lower, 40 °C or lower, 35 °C or lower, 30 °C or lower, 25 °C or lower), the signal emitted by the reporter group on the detection probe is monitored, thereby obtaining a curve of the signal intensity of the reporter group that varies with temperature changes. The cooling rate can be determined conventionally by those skilled in the art. For example, the cooling rate can be as follows: the temperature is decreased by 0.01 °C to 1 °C (e.g., 0.01 °C to 0.05 °C, 0.05 °C to 0.1 °C, 0.1 °C to 0.5 °C, 0.5 °C to 1 °C, 0.04 °C to 4 °C, e.g., 0.01 °C, 0.02 °C, 0.03 °C, 0.04 °C, 0.05 °C, 0.06 °C, 0.07 °C, 0.08 °C, 0.09 °C, 0.1 °C, 0.2 °C, 0.3 °C, 0.4 °C, 0.5 °C, 0.6 °C, 0.7 °C, 0.8 °C, 0.9 °C or 1.0 °C) for each step, and each step is maintained for 0.5 seconds to 15 seconds (e.g., 0.5 seconds to 1 second, 1 second to 2 seconds, 2 seconds to 3 seconds, 3 seconds to 4 seconds, 4 seconds to 5 seconds, 5 seconds to 10 seconds, 10 seconds to 15 seconds), or the temperature is decreased by 0.01 °C to 1 °C (e.g., 0.01 °C to 0.05 °C, 0.05 °C to 0.1 °C, 0.1 °C to 0.5 °C, 0.5 °C to 1 °C, 0.04 °C to 0.4 °C, e.g., 0.01 °C, 0.02 °C, 0.03 °C, 0.04 °C, 0.05 °C, 0.06 °C, 0.07 °C, 0.08 °C, 0.09 °C, 0.1 °C, 0.2 °C, 0.3 °C, 0.4 °C, 0.5 °C, 0.6 °C, 0.7 °C, 0.8 °C, 0.9 °C, or 1.0 °C) per second.

[0084] Subsequently, the obtained curve can be differentiated to obtain the melting curve of the product of step (2). According to the melting peak (melting point) of the melting curve, the presence of the target nucleic acid corresponding to the melting peak (melting point) can be determined.

[0085] In the method of the present invention, the detection probes used can each independently use the same or different reporter groups. In a certain preferred embodiment, the detection probes used have the same reporter group. In this case, melting curve analysis can be performed on the product of (2), and the presence of a specific target nucleic acid can be determined according to the melting peak (melting point) of the melting curve. In a certain preferred embodiment, the detection probes used have different reporter groups. In this case, when the product of step (2) is subjected to melting curve analysis, the signals of each reporter group can be monitored in real time, whereby a plurality of melting curves corresponding to the signal of one reporter group can be obtained. Then, the presence of a specific target nucleic acid can be determined according to the signal species of the reporter group and the melting peak (melting point) of the melting curve. Therefore, the method of the present invention can achieve simultaneous detection (multiplex detection) of one or more (for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) target nucleic acid sequences.

[0086] Without being bound by theoretical limitations, for the same reporter group (same melting curve), the resolution or accuracy of melting curve analysis can reach 0.5 °C or more. In other words, in melting curve analysis, two melting peaks with a difference in melting points of the same melting curve of less than 0.5 °C (for example, 0.1 °C, 0.2 °C, 0.3 °C, 0.4 °C, 0.5 °C) can be distinguished. Therefore, in certain embodiments of the method of the present invention, the melting point difference between various duplexes formed by various target nucleic acids and their detection probes can be at least 0.5 °C using the same reporter group, so that different duplexes (and thus different target nucleic acids) can be distinguished and identified by melting curve analysis. However, in order to facilitate distinction and identification, in some cases, it is preferable that the difference in melting points between the two duplexes is larger. Therefore, in certain embodiments of the method of the present invention, the difference in melting points between two duplexes can be any desired value (for example, at least 0.5 °C, at least 1 °C, at least 2 °C, at least 3 °C, at least 4 °C, at least 5 °C, at least 8 °C, at least 10 °C, at least 15 °C, or at least 20 °C) as long as the melting point difference can be distinguished and identified by melting curve analysis.

[0087] In certain preferred embodiments, steps (1) to (3) of the method of the present invention are the following steps (a) to (g): (a) Providing a sample containing one or more target nucleic acids, providing a universal primer, and for each target nucleic acid to be amplified, providing a target-specific primer pair and a detection probe, wherein the universal primer, the target-specific primer pair, and the detection probe are as defined above; (b) Mixing the sample with the universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase (for example, a template-dependent nucleic acid polymerase; for example, a DNA polymerase, particularly a heat-resistant DNA polymerase); (c) Incubating the product of the previous step under conditions capable of nucleic acid denaturation; (d) incubating the product of the previous step under conditions that allow nucleic acid annealing or hybridization; (e) incubating the product of the previous step under conditions that allow nucleic acid extension; (f) optionally, repeating steps (c) to (e) one or more times; (g) performing melting curve analysis on the product of the previous step. It can be carried out by a protocol comprising:

[0088] Steps (a) to (g) have been described in detail above.

[0089] Primer Sets and Kits In one aspect, the present invention provides a primer set comprising a universal primer and one or more target-specific primer pairs, wherein the universal primer comprises a first universal sequence, each target-specific primer pair can amplify a target nucleic acid and comprises a forward primer and a reverse primer, the forward primer comprising a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid, the forward nucleotide sequence being located at the 3'-end of the second universal sequence, the reverse primer comprising the first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, the reverse nucleotide sequence being located at the 3'-end of the first universal sequence, and under conditions allowing nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence, there is a difference between the second universal sequence and the first universal sequence, and one or more nucleotides located at the 3'-end of the first universal sequence are each independently deleted or substituted, and the first universal sequence must not be completely complementary to the complementary sequence of the forward primer.

[0090] In certain preferred embodiments, the primer set comprises from 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target-specific primer pairs, for example at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target-specific primer pairs.

[0091] It will be readily understood that such primer sets can be used to carry out the methods of the invention described in detail above. Accordingly, the various technical features described in detail above for the universal primer, the target-specific primer pair, the target nucleic acid, and the sample can also be applied to the technical solutions including the primer sets of the present application. Accordingly, in certain preferred embodiments, the primer set comprises a universal primer and / or a target-specific primer pair as defined above.

[0092] In certain preferred embodiments, one target nucleic acid in a sample can be amplified using a primer set, the primer set comprising a universal primer and a first target-specific primer pair comprising a first forward primer and a first reverse primer, The universal primer comprises a first universal sequence, The first forward primer includes a second universal sequence and a first forward nucleotide sequence specific to the first target nucleic acid, the first forward nucleotide sequence is located at the 3'-end of the second universal sequence, and under the conditions of nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence, there is a difference between the second universal sequence and the first universal sequence, and the difference includes that one or more nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted. The first reverse primer includes a first universal sequence and a first reverse nucleotide sequence specific to the first target nucleic acid, and the first reverse nucleotide sequence is located at the 3'-end of the first universal sequence. The first forward primer and the first reverse primer can specifically amplify the first target nucleic acid. The first universal sequence is not completely complementary to the complementary sequence of the first forward primer.

[0093] In certain preferred embodiments, the primer set can be used to amplify two target nucleic acids in a sample. The primer set includes a universal primer, a first target-specific primer pair, and a second target-specific primer pair. The universal primer is as defined above. The first target-specific primer pair includes a first forward primer and a first reverse primer capable of amplifying the first target nucleic acid. The second target-specific primer pair includes a second forward primer and a second reverse primer capable of amplifying the second target nucleic acid. The first forward primer, the first reverse primer, the second forward primer, and the second reverse primer are as defined above. Similarly, in certain preferred embodiments, the primer set of the present invention can be used to amplify three or more target nucleic acids in a sample. The primer set includes a universal primer and three or more target-specific primer pairs capable of amplifying three or more target nucleic acids.

[0094] Furthermore, for convenience, the primer set of the present invention can be combined with one or more reagents necessary for carrying out the method (amplification method or detection method) of the present invention to prepare a kit. It will be readily understood that such a kit can be used to carry out the method of the present invention described in detail above. Therefore, the various technical features detailed above for the various components are equally applicable to the various components of the kit. Furthermore, such a kit may also include other reagents necessary for carrying out the method of the present invention.

[0095] Accordingly, in another aspect, the present application provides a kit comprising the primer set described above and one or more components selected from the group consisting of a nucleic acid polymerase, a reagent for nucleic acid amplification, a reagent for sequencing, a reagent for gene chip detection, a reagent for melting curve analysis, or any combination thereof.

[0096] In certain preferred embodiments, the nucleic acid polymerase is a template-dependent nucleic acid polymerase such as a DNA polymerase, particularly a heat-resistant DNA polymerase. In certain preferred embodiments, the nucleic acid polymerase is as defined above.

[0097] Reagents for nucleic acid amplification can be determined by those skilled in the art according to conventional methods and are not limited, but include, for example, an action buffer for an enzyme (e.g., nucleic acid polymerase), dNTP (labeled or unlabeled), water, a solution containing ions (e.g., Mg 2+ ), a single-stranded DNA-binding protein (SSB), or any combination thereof.

[0098] Reagents for sequencing can be determined by those skilled in the art according to conventional methods and are not limited, but include, for example, an action buffer for an enzyme (e.g., nucleic acid polymerase), dNTP (labeled or unlabeled), ddNTP (labeled or unlabeled), water, a solution containing ions (e.g., Mg 2+ ), a single-stranded DNA-binding protein (SSB), ligase, nucleic acid linker, sequencing primer, or any combination thereof.

[0099] Reagents for gene chip detection can be determined by those skilled in the art according to conventional methods and are not limited, but include, for example, an action buffer for an enzyme (e.g., nucleic acid polymerase), dNTP (labeled or unlabeled), water, hybridization buffer, washing buffer, labeling reagent, or any combination thereof.

[0100] Reagents for melting curve analysis can be determined by those skilled in the art according to conventional methods and are not limited, but include detection probes. In certain preferred embodiments, the detection probe is a self-quenching probe, for example, a self-quenching fluorescent probe. In certain preferred embodiments, the detection probe is as defined above.

[0101] Based on the principles described in detail in this application, those skilled in the art can modify, replace, or combine various technical features of the technical solution of the present invention without departing from the spirit and scope of the present invention. All such technical solutions and their modifications are included within the scope of the claims of this application or their equivalents.

Advantages of the Invention

[0102] Beneficial Effects of the Present Invention Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) Compared with the conventional HAND system and the amplification method using the conventional HAND system, the technical solution of the present invention can achieve simultaneous asymmetric amplification (multiple asymmetric amplification) of multiple target nucleic acids. (2) Compared with the conventional multiple asymmetric PCR amplification method, the technical solution of the present invention can effectively suppress the non-specific amplification of primer dimers and significantly improve the specificity and detection sensitivity of amplification.

[0103] Therefore, the present invention can simultaneously and asymmetrically amplify multiple target nucleic acids, simultaneously achieve effective multiple PCR amplification and asymmetric PCR amplification, and develop a new method that can meet the clinical requirements for simultaneously amplifying and detecting multiple target nucleic acids.

[0104] Embodiments of the present invention will be described in detail below with reference to the drawings and examples. Those skilled in the art will understand that the following drawings and examples are not intended to limit the scope of the present invention, but are only used to illustrate the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the detailed description of the following preferred embodiments.

Brief Description of the Drawings

[0105]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0106] Specific model for implementing the present invention Here, the present invention will be described with reference to the following examples, which are intended to illustrate the present invention but not to limit it thereto. It should be understood that these examples are only used to explain the principles and technical effects of the present invention, but do not represent all possibilities of the present invention. The present invention is not limited to the materials, reaction conditions, or parameters mentioned in these examples. Those skilled in the art can implement other technical solutions using other similar materials or reaction conditions according to the principles of the present invention. Such technical solutions do not deviate from the basic principles and concepts described in the present invention and are included in the scope of the present invention.

Example

[0107] Example 1 In this example, by using a DNA fragment containing the gene polymorphism site rs2252992 on human chromosome 21 as the target nucleic acid to be amplified, the HAND system, the conventional asymmetric PCR system, and the system (primer set) of the present invention were examined to produce single-stranded nucleic acid products. The sequences of the primers and probes used in this example are shown in Table 1. The instrument used in this example was a SLAN 96 real-time fluorescence PCR machine (Xiamen Zeesan Biotech Co., Ltd., Xiamen).

[0108] Briefly, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included 1× Taq PCR buffer (TaKaRa, Beijing), 5.0 mM MgCl2, 0.2 mM dNTP, 1 U Taq DNA polymerase (TaKaRa, Beijing), 0.2 μM rs2252992-P probe, 5 μL of human genomic DNA (the rs2252992 genotype was A / A homozygous) or negative control (water), and primers. (1) To the HAND system, 0.03 μM rs2252992-F1, 0.03 μM rs2252992-R, and 0.3 μM Tag primer (i.e., universal primer) were added. (2) 0.03 μM of rs2252992-F1 and 0.3 μM of rs2252992-R were added to the conventional asymmetric PCR system, (3) 0.03 μM of rs2252992-F2, 0.03 μM of rs2252992-R, and 0.3 μM of Tag primer were added to the PCR system based on the method of the present invention.

[0109] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 minutes, 10 cycles of (denaturation at 95°C for 15 seconds, annealing at 65°C to 56°C for 15 seconds (decreasing by 1°C per cycle), extension at 76°C for 20 seconds), and 50 cycles of (denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 76°C for 20 seconds). The fluorescence signal of the CY5 channel was collected during the annealing stage. After PCR amplification, melting curve analysis was performed based on the following program: denaturation at 95°C for 1 minute; incubation at 37°C for 3 minutes; then, the temperature was raised from 40°C to 85°C at a heating rate of 0.04°C / second, and the fluorescence signal of the CY5 channel was collected. Finally, each PCR product was analyzed by 2% agarose gel electrophoresis. The experimental results are shown in Figures 2 to 4.

[0110]

Table 1

[0111] Figure 2 shows the results of real-time PCR amplification using the HAND system, the conventional asymmetric PCR system, and the system of the present invention in Example 1. In the figure, the black and gray dashed lines respectively represent the amplification curves of human genomic DNA and negative control amplified using the HAND system, the black and gray dotted lines respectively represent the amplification curves of human genomic DNA and negative control amplified using the conventional asymmetric PCR system, and the black and gray solid lines respectively represent the amplification curves of human genomic DNA and negative control amplified using the system of the present invention. The results showed that all three systems could effectively and specifically amplify human genomic DNA and generate corresponding amplification signals (black dashed line, black dotted line, and black solid line), and there were no amplification signals in the negative controls of each system (gray dashed line, gray dotted line, and gray solid line). Furthermore, it was noted that the amplification curve of the conventional asymmetric PCR system (black dotted line) had the minimum Ct value caused by direct amplification of the target nucleic acid by a high concentration of target-specific primers.

[0112] Figure 3 shows the results of melting curve analysis after amplification using the HAND system, the conventional asymmetric PCR system, and the system of the present invention in Example 1. In the figure, the black and gray dashed lines respectively represent the results of melting curve analysis after amplifying human genomic DNA and negative control using the HAND system, the black and gray dotted lines respectively represent the results of melting curve analysis after amplifying human genomic DNA and negative control using the conventional asymmetric PCR system, and the black and gray solid lines respectively represent the results of melting curve analysis after amplifying human genomic DNA and negative control using the system of the present invention.

[0113] Figure 4 shows the results of agarose gel electrophoresis of amplification products obtained using the HAND system, a conventional asymmetric PCR system, and the system of the present invention in Example 1. In the figure, lane M represents the molecular weight marker, and lanes 1 to 3 represent the products obtained by amplifying human genomic DNA using the HAND system (lane 1), the system of the present invention (lane 2), and the conventional asymmetric PCR system (lane 3), respectively. Lanes 4 to 6 represent the products obtained by amplifying the negative control using the HAND system, the system of the present invention, and the conventional asymmetric PCR system, respectively.

[0114] The results of FIGS. 3 and 4 show that when the HAND system was used for amplification, the amplification products were basically double-stranded nucleic acids and single-stranded nucleic acid products could not be generated (FIG. 4, lane 1). Therefore, during the melting curve analysis process, the probe could not effectively hybridize to the amplification products and could not generate an effective melting peak (FIG. 3, black dashed line). Therefore, when the HAND system was used for amplification, efficient probe melting curve analysis could not be performed on the amplification products. However, when the system of the present invention and the conventional asymmetric PCR system were used for amplification, a large amount of single-stranded nucleic acid products were generated (FIG. 4, lanes 2 and 3). Therefore, during the melting curve analysis process, the probe could efficiently hybridize to the amplification products and generate specific melting peaks (FIG. 3, black solid line and black dotted line). Furthermore, the results of FIGS. 3 and 4 show that none of the negative control systems using water as a template could generate effective melting peaks (FIG. 3, gray dashed line, gray dotted line, and gray solid line), which indicates that the desired amplification products were not generated during the PCR process (FIG. 4, lanes 4 to 6).

[0115] The results of this example demonstrated that the system of the present invention can be used to obtain asymmetric amplification of target nucleic acids and can therefore be used in combination with probe melting curve analysis.

[0116] Example 2 In this example, a DNA fragment of the gene polymorphism site rs2252992 on human chromosome 21 was used as the target nucleic acid to be amplified, and the effect of the difference between the second universal sequence and the first universal sequence (i.e., different variant types of the second universal sequence with respect to the first universal sequence) on asymmetric amplification was examined. The sequences of the primers and probes used in this example are shown in Table 2. The nucleotide at the 3'-end of the universal primer (Tag primer) used here is A. Five types of forward primers were designed. That is, the nucleotide at the 3'-end of the second universal sequence of rs2252992-F-C is C, the nucleotide at the 3'-end of the second universal sequence of rs2252992-F-G is G, the nucleotide at the 3'-end of the second universal sequence of rs2252992-F-T is T, the nucleotide at the 3'-end of the second universal sequence of rs2252992-F-A is A, and the second nucleotide from the last of the 3'-end of the second universal sequence of rs2252992-F-D lacks G. rs2252992-F-C, rs2252992-F-G, rs2252992-F-T and rs2252992-F-D were respectively used in the system of the present invention, and their complementary sequences formed mismatches A-G, A-C, A-A and GA-TA with the universal primer during amplification. The control primer rs2252992-F-A was used in the HAND system, and its complementary sequence completely matched the universal primer during amplification. The equipment used in this example was a SLAN 96 real-time fluorescence PCR machine.

[0117] Briefly described, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included 1× Taq PCR buffer, 5.0 mM MgCl2, 0.2 mM dNTP, 1 U Taq DNA polymerase, 0.4 μM rs2252992-P probe, 0.04 μM rs2252992-R primer, 1.6 μM Tag primer, 5 μL of human genomic DNA (the rs2252992 genotype was T / C heterozygous) or negative control (water), and 0.04 μM of the designated forward primer (i.e., rs2252992-F-A, or rs2252992-F-C, or rs2252992-F-G, or rs2252992-F-T, or rs2252992-F-D primer).

[0118] The PCR amplification program was a pre-denaturation at 95°C for 5 minutes, 10 cycles of (denaturation at 95°C for 15 seconds, annealing at 65°C to 56°C for 15 seconds (decreasing 1°C per cycle), extension at 76°C for 20 seconds), and 50 cycles of (denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 76°C for 20 seconds). After the PCR amplification was completed, melting curve analysis was performed, and its program was as follows: denaturation at 95°C for 1 minute; incubation at 37°C for 3 minutes; then, melting curve analysis was performed at a temperature rising from 50°C to 85°C at a heating rate of 0.04°C / second, and the fluorescence signal of the CY5 channel was collected. The results of the melting curve analysis are shown in FIG. 5.

[0119]

Table 2

[0120] FIG. 5 shows the results of melting curve analysis after amplification using different forward primers in Example 2. In the figure, the gray dashed line, black solid line, black dashed line, gray solid line, or black dotted line represents the results of melting curve analysis after amplification using rs2252992-F-A, rs2252992-F-C, rs2252992-F-G, rs2252992-F-T, or rs2252992-F-D, respectively.

[0121] The experimental results in Figure 5 showed that when the rs2252992-F-A primer (which had a completely matching complementary sequence with the universal primer) was used for amplification, the entire reaction system was equivalent to the HAND system, and the amplification product did not contain single-stranded nucleic acid products. Therefore, no target melting peak was observed in the melting curve (gray dashed line). In contrast, when the primers of rs2252992-F-C (black solid line), rs2252992-F-G (black dashed line), rs2252992-F-T (gray solid line), or rs2252992-F-D (black dotted line) were used for amplification, since the universal primer was not completely complementary to the complementary sequence of the forward primer (A-G, A-C, A-A, or GA-TA mismatches were formed at the 3'-end of the universal primer), the amplification efficiencies of the two nucleic acid strands were different, and asymmetric amplification was formed, thereby obtaining single-stranded nucleic acid products. Correspondingly, a target melting peak was observed in the melting curve of the amplification product, indicating this. This experimental result shows that various mismatches / substitutions were applicable to the system of the present invention. It is also worth noting that different mismatch types may contribute to differences in amplification efficiency (reflected by the difference in the height of the melting peak). Therefore, when using the system of the present invention for asymmetric amplification, an appropriate mismatch type can be selected in the forward primer / second universal sequence according to the actual needs.

[0122] Example 3 In this example, taking the typing of gene polymorphism sites rs2252992 and rs4816597 as an example, it was demonstrated that the system of the present invention could achieve double asymmetric amplification in a single reaction tube and could be used for melting curve analysis of probes. The sequences of the primers and probes used in this example are shown in Table 3. The instrument used in this example was the SLAN 96 real-time fluorescence PCR machine.

[0123] Briefly described, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system contained 1× Taq PCR buffer, 5.0 mM MgCl2, 0.2 mM dNTP, 1 U Taq DNA polymerase, 0.05 μM rs4816597-F, 0.05 μM rs4816597-R, 0.4 μM rs4816597-P, 0.04 μM rs2252992-F, 0.04 μM rs2252992-R, 0.4 μM rs2252992-P, 1.6 μM Tag primer, 5 μL of human genomic DNA, or a negative control (water). In this example, four samples were detected (Sample 1, Sample 2, Sample 3, and Sample 4; one sample was detected using each PCR reaction system). The genotypes of the rs2252992 and rs4816597 sites in Sample 1 were determined to be T / C and C / C, the genotypes of the rs2252992 and rs4816597 sites in Sample 2 were determined to be T / C and T / C, the genotypes of the rs2252992 and rs4816597 sites in Sample 3 were determined to be T / T and C / C, and the genotypes of the rs2252992 and rs4816597 sites in Sample 4 were determined to be C / C and C / C.

[0124] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 minutes; 10 cycles of (denaturation at 95°C for 15 seconds, annealing at 65°C to 56°C for 15 seconds (decreasing 1°C per cycle), extension at 76°C for 20 seconds); 50 cycles of (denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 76°C for 20 seconds); then, melting curve analysis was performed, and the program was as follows: denaturation at 95°C for 1 minute, incubation at 37°C for 3 minutes, and then melting curve analysis was performed at a temperature rising from 40°C to 85°C at a heating rate of 0.04°C / second, and the fluorescence signal of the CY5 channel was collected. The results of the melting curve analysis are shown in Figure 6.

[0125]

Table 3

[0126] Figure 6 shows the results of melting curve analysis after amplification using the system of the present invention in Example 3. In the figure, the solid black line (Sample 1), the dashed black line (Sample 2), the solid gray line (Sample 3), and the dashed gray line (Sample 4) represent the results of melting curve analysis after using the system of the present invention to amplify Samples 1 to 4, respectively.

[0127] The results in Figure 6 show that the genotypes of the gene polymorphic sites rs2252992 and rs4816597 in Sample 1 (solid black line) are T / C and C / C, respectively; the genotypes of the gene polymorphic sites rs2252992 and rs4816597 in Sample 2 (dashed black line) are T / C and T / C, respectively; the genotypes of the gene polymorphic sites rs2252992 and rs4816597 in Sample 3 (solid gray line) are T / T and C / C, respectively; the genotypes of the gene polymorphic sites rs2252992 and rs4816597 in Sample 4 (dashed gray line) are C / C and C / C, respectively; there is no melting peak in the negative control (solid gray line), and the genotyping results of each sample were consistent with the results obtained by sequencing. These results indicate that the system of the present invention can simultaneously and asymmetrically amplify two target nucleic acids in a single reaction system, and can generate sufficient single-stranded nucleic acid products for effective and reliable melting curve analysis, thereby realizing the identification of two target nucleic acids (for example, genotyping of two gene polymorphic sites). Therefore, by combining with melting curve analysis technology, the system of the present invention was able to simultaneously achieve the detection and identification (for example, genotyping) of two target nucleic acids.

[0128] Example 4 In this example, eight gene polymorphism sites (i.e., rs979393, rs34521064, rs2835906, rs7275547, rs418298, rs60871880, rs4816597, and rs2252992) were taken as examples to demonstrate that the system of the present invention can achieve 8-plex asymmetric amplification in a single reaction tube and can be used for melting curve analysis of probes. The sequences of the primers and probes used in this example are shown in Table 4. The instrument used in this example was the SLAN 96 real-time fluorescence PCR machine.

[0129] Briefly, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included 1× Taq PCR buffer, 5.0 mM MgCl2, 0.24 mM dNTP, 1 U Taq DNA polymerase, 5 μL of human genomic DNA, primers, and probes. The concentrations of the primers and probes used are shown in Table 4. In this example, a total of six samples were detected (Samples 5 to 10, with each PCR reaction system detecting one sample).

[0130] The PCR amplification program was: pre-denaturation at 95°C for 5 minutes; 4 cycles of (denaturation at 95°C for 15 seconds, annealing at 52°C for 15 seconds, extension at 76°C for 20 seconds); 55 cycles of (denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 76°C for 20 seconds). Fluorescence signals in the FAM, HEX, ROX, and CY5 channels were collected during the annealing stage. Then, melting curve analysis was performed, and the program was as follows: denaturation at 95°C for 1 minute and incubation at 37°C for 3 minutes; then, the temperature was raised from 40°C to 85°C at a heating rate of 0.04°C / second, and fluorescence signals in the FAM, HEX, ROX, and CY5 channels were collected. The experimental results are shown in Figure 7.

[0131]

Table 4

[0132] Figure 7 shows the results of post-amplification melting curve analysis using the system of the present invention in Example 4. In the figure, the solid black line (Sample 5), the dashed black line (Sample 6), the dotted black line (Sample 7), the solid gray line (Sample 8), the dashed gray line (Sample 9), and the dotted gray line (Sample 10) respectively represent the results of melting curve analysis after amplifying Samples 5 to 10 using the system of the present invention.

[0133]

Table 5

[0134] Furthermore, the genotyping results of 8 gene polymorphism sites in Samples 5 to 10 were also identified by sequencing. The results showed that the genotyping results of each sample obtained by the system of the present invention (Figure 7 and Table 5) were completely consistent with the results obtained by sequencing.

[0135] These results indicate that the system of the present invention can simultaneously and asymmetrically amplify 8 target nucleic acids in a sample in a single reaction system, generating single-stranded nucleic acid products sufficient for effective and reliable melting curve analysis, thereby enabling the identification of multiple target nucleic acids (e.g., genotyping of multiple gene polymorphism sites). Therefore, in combination with melting curve analysis technology, the system of the present invention can simultaneously achieve the detection and identification (e.g., genotyping) of multiple target nucleic acids.

[0136] Example 5 In this example, to compare the analysis sensitivity between the system of the present invention and the conventional multiplex asymmetric PCR system, genotyping of samples containing human genomic DNA at different concentrations was taken as an example. The genomic DNA used in this example had eight polymorphic sites of known genotypes, specifically rs979393: T / G; rs34521064: T / C; rs2835906: T / C; rs7275547: G / C; rs1005546: C / C; rs857998: C / C; rs4816597: T / C; rs2252992: C / C). The sequences of the primers and probes used in this example are shown in Table 6. The instrument used in this example was the SLAN 96 real-time fluorescence PCR machine.

[0137] Briefly, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included 1× Taq PCR buffer, 5.0 mM MgCl2, 0.24 mM dNTP, 2 U Taq DNA polymerase, 5 μL of human genomic DNA (at concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL, 0.01 ng / μL, or 0.005 ng / μL), and primers and probes. The concentrations of the primers and probes used are shown in Table 6.

[0138] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 minutes; 6 cycles of (denaturation at 95°C for 15 seconds, annealing at 52.5°C for 15 seconds, extension at 76°C for 20 seconds); 55 cycles of (denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 76°C for 20 seconds); fluorescence signals of the FAM, HEX, ROX, and CY5 channels were collected during the annealing stage. Then, melting curve analysis was performed, and its program was as follows: denaturation at 95°C for 1 minute and incubation at 37°C for 3 minutes; then, the temperature was raised from 40°C to 85°C at a heating rate of 0.04°C / second, and fluorescence signals of the FAM, HEX, ROX, and CY5 channels were collected. The experimental results are shown in FIGS. 8 and 9.

[0139]

Table 6

[0140] Figure 8 shows the results of post-amplification melting curve analysis using the system of the present invention in Example 5. In the figure, the black dotted line, black dashed line, gray dotted line, gray dashed line, black solid line, and gray solid line represent the results of melting curve analysis after amplifying samples with DNA concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL, 0.01 ng / μL, or 0.005 ng / μL, respectively.

[0141] Figure 9 shows the results of post-amplification melting curve analysis using a conventional multiplex asymmetric PCR system in Example 5. In the figure, the black dotted line, black dashed line, gray dotted line, gray dashed line, black solid line, and gray solid line represent the results of melting curve analysis after amplifying samples with genomic DNA concentrations of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.05 ng / μL, 0.01 ng / μL, or 0.005 ng / μL, respectively.

[0142] The results of Figure 8 indicate that even when the concentration of human genomic DNA was as low as 0.05 ng / μL (gray dashed line), the system of the present invention could stably and accurately detect the genotypes of all 8 gene polymorphism sites. In contrast, the results of Figure 9 show that with the conventional multiplex asymmetric PCR system, all genotypes of the 8 gene polymorphism sites could be detected only when the concentration of human genomic DNA was 10 ng / μL (black dotted line) and 1 ng / μL (black dashed line). When the concentration of human genomic DNA was 0.1 ng / μL (gray dotted line), the genotypes of some gene polymorphism sites (e.g., rs34521064 site, rs4816597 site) could not be detected and discriminated (no distinguishable melting peaks were generated). This is presumably because in the conventional multiplex asymmetric PCR reaction system, multiple types of high-concentration primers and probes interacted with each other, and the asymmetric amplification of some gene polymorphism sites could not be effectively carried out, resulting in an inability to generate a sufficient amount of single-stranded products for melting curve analysis.

[0143] The above results indicated that the detection sensitivity of the system of the present invention was significantly higher than that of the conventional multiplex asymmetric PCR system. This is mainly because the system of the present invention uses low-concentration target-specific primers and high-concentration universal primers for amplification, so the interference between primers is effectively reduced, the non-specific amplification of dimers, etc. is reduced, the amplification of each target nucleic acid in the reaction system reaches equilibrium, and thereby the detection sensitivity of the entire reaction system is improved.

[0144] Although specific embodiments of the present invention are described in detail, those skilled in the art can make various modifications and changes in detail in light of all the teachings disclosed, and understand that all these changes are within the scope of the present invention. The full scope of the present invention is given by the appended claims and equivalents thereof.

Claims

1. A method for amplifying one or more target nucleic acids in a sample, comprising: (1) providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair for each target nucleic acid to be amplified, wherein the universal primer contains a first universal sequence, and the first universal sequence is located at the 3'-portion of the universal primer or constitutes the 3'-portion of the universal primer; the target-specific primer pair can amplify the target nucleic acid, and includes a forward primer and a reverse primer, wherein the forward primer contains a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid, the forward nucleotide sequence is located at the 3'-end of the second universal sequence, and the forward nucleotide sequence is located at the 3'-portion of the forward primer or constitutes the 3'-portion of the forward primer; the reverse primer contains the first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, the reverse nucleotide sequence is located at the 3'-end of the first universal sequence, and the reverse nucleotide sequence is located at the 3'-portion of the reverse primer or constitutes the 3'-portion of the reverse primer; under conditions allowing nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence, there is a difference between the second universal sequence and the first universal sequence, and the difference includes that one or more nucleotides located at the 3'-end of the first universal sequence are independently deleted or substituted, and the first universal sequence is not completely complementary to the complementary sequence of the forward primer; (2) under conditions allowing nucleic acid amplification, using the universal primer and the target-specific primer pair to amplify the target nucleic acid in the sample via a PCR reaction; A method comprising the above steps.

2. The method is as follows: (1) The method is used for amplifying 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target nucleic acids; In step (1) of the method, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target-specific primer pairs are provided; In step (2) of the method, the universal primer has an effective concentration higher than the effective concentrations of the forward primer and the reverse primer; In step (2) of the method, the forward primer and the reverse primer have the same or different effective concentrations; The sample or target nucleic acid contains mRNA, and a reverse transcription reaction is performed on the sample before step (2) of the method is carried out; and In step (2) of the method, a PCR reaction is performed using a nucleic acid polymerase. The method according to claim 1, having one or more technical features selected from the above.

3. The method is as follows: In step (1) of the method, the universal primer has an effective concentration 1 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 50 times or more higher than the effective concentrations of the forward primer and the reverse primer; In step (2), the nucleic acid polymerase is a template-dependent nucleic acid polymerase; In step (3), the nucleic acid polymerase is a DNA polymerase; In step (4), the nucleic acid polymerase is a thermostable DNA polymerase. (5) the nucleic acid polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus litoralis, Thermus antiranildanni, Thermus caldophilus, Thermus clearophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus osimai, Thermus rubber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermofilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus and Aquifex aeolicus; and, (6) the nucleic acid polymerase is Taq polymerase, The method according to claim 2, having one or more technical features selected from the group consisting of

4. The method is as follows: (1) the universal primer consists of the first universal sequence or includes the first universal sequence and an additional sequence, and the additional sequence is located at the 5' end of the first universal sequence; (2) the universal primer has a length of 5 nt to 15 nt, 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, or 40 nt to 50 nt; and, (3) the universal primer or any of its components contains or consists of naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof, The method according to claim 1, having one or more technical features selected from the group consisting of

5. The method is as follows: (1) in the forward primer, the forward nucleotide sequence is directly linked to the 3' end of the second universal sequence or is linked to the 3' end of the second universal sequence via a nucleotide linker; (2) The forward primer further includes an additional sequence located at the 5'-end of the second universal sequence; (3) The forward primer contains or consists of, from 5' to 3', the second universal sequence and the forward nucleotide sequence; or contains or consists of, from 5' to 3', the second universal sequence, a nucleotide linker, and the forward nucleotide sequence; or contains or consists of, from 5' to 3', an additional sequence, the second universal sequence, and the forward nucleotide sequence; or contains or consists of, from 5' to 3', an additional sequence, the second universal sequence, a nucleotide linker, and the forward nucleotide sequence; (4) The forward nucleotide sequence has a length of 10 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt; (5) The forward primer has a length of 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, 100 nt to 110 nt; (6) The forward primer or any of its components contains or consists of naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof; (7) In the reverse primer, the reverse nucleotide sequence is directly linked to the 3'-end of the first universal sequence, or the reverse nucleotide sequence is linked to the 3'-end of the first universal sequence via a nucleotide linker; (8) The reverse primer further includes an additional sequence located at the 5'-end of the first universal sequence; (9) The reverse primer contains, or consists of, the first universal sequence and a reverse nucleotide sequence from 5' to 3'; or contains, or consists of, the first universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'; or contains, or consists of, an additional sequence, the first universal sequence, and a reverse nucleotide sequence from 5' to 3'; or contains, or consists of, an additional sequence, the first universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'; (10) The reverse nucleotide sequence has a length of 10 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, or 70 nt to 80 nt; (11) The reverse primer has a length of 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, or 100 nt to 110 nt; (12) The reverse primer or any of its components contains, or consists of, naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (13) The first universal sequence is not completely complementary to the complementary sequence of the forward primer; and, (14) The difference between the second universal sequence and the first universal sequence includes that 1 to 5, 5 to 10, 10 to 15, 15 to 20, or more nucleotides at the 3' end of the first universal sequence are independently deleted or substituted, or the difference lies therein, The method according to claim 1, having one or more technical features selected from the above.

6. The method is as follows: (1) The nucleotide linker contains 1 to 5, 5 to 10, 10 to 15, 15 to 20, or more nucleotides; (2) The additional sequence contains 1 to 5, 5 to 10, 10 to 15, 15 to 20, or more nucleotides; (3) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; and, (4) One to five, five to ten, ten to fifteen, fifteen to twenty or more nucleotides at the 3' end of the first universal array are not complementary to the complementary sequence of the forward primer; The method according to claim 5, having one or more technical features selected from:

7. The method is as follows: (1) The sample contains DNA, RNA, or any combination thereof; (2) The target nucleic acid to be amplified is DNA, RNA, or any combination thereof; (3) The target nucleic acid to be amplified is single-stranded or double-stranded; and (4) The sample or target nucleic acid is obtained from prokaryotes, eukaryotes or viruses or viroids, The method according to claim 1, having one or more technical features selected from:

8. The method is as follows: (1) The DNA is genomic DNA or cDNA; (2) The RNA is mRNA; (3) The eukaryote is selected from protozoa, parasites, fungi, yeast, plants, and animals; (4) The eukaryote is a mammal; (5) The eukaryote is a human; and (6) The virus is selected from herpes virus, HIV, influenza virus, EBV, hepatitis virus, and poliovirus; The method according to claim 7, having one or more technical features selected from:

9. Steps (1) and (2) of the method are the following steps (a) to (f): (a) Providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair for each target nucleic acid to be amplified, The step in which the universal primer and the target-specific primer pair are as defined in claim 1; (b) Mixing the sample with the universal primer, the target-specific primer pair, and a nucleic acid polymerase; (c) Incubating the product of the previous step under conditions capable of nucleic acid denaturation; (d) Incubating the product of the previous step under conditions capable of nucleic acid annealing or hybridization; (e) Incubating the product of the previous step under conditions capable of nucleic acid elongation; (f) Repeating step (c) to step (e) either not at all or one or more times; The method according to claim 1, which is carried out by a protocol comprising:

10. The method is as follows: (1) In step (c), incubate the product of step (b) at a temperature of 80°C to 105°C, thereby enabling said nucleic acid denaturation; (2) In step (c), incubate the product of step (b) for 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, or 2 minutes to 5 minutes; (3) In step (d), incubate the product of step (c) at a temperature of 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 60°C to 65°C, or 65°C to 70°C, thereby enabling said nucleic acid annealing or hybridization; (4) In step (d), incubate the product of step (c) for 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, or 2 minutes to 5 minutes; (5) In step (e), incubate the product of step (d) at a temperature of 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 60°C to 65°C, 65°C to 70°C, 70°C to 75°C, 75°C to 80°C, 80°C to 85°C, thereby enabling said nucleic acid elongation; (6) In step (e), incubate the product of step (d) for 10 seconds to 20 seconds, 20 seconds to 40 seconds, 40 seconds to 60 seconds, 1 minute to 2 minutes, 2 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, or 20 minutes to 30 minutes; (7) Perform steps (d) and (e) at the same temperature or different temperatures; and, (8) Repeat steps (c) to (e) at least once, and optionally, when repeating steps (c) to (e) more than once, the conditions used in steps (c) to (e) of each cycle are independently the same or different, The method according to claim 9, having one or more technical features selected from the above.

11. A method for detecting one or more target nucleic acids in said sample, comprising: (i) a step of amplifying one or more target nucleic acids in said sample using the method according to claim 10; and (ii) a step of performing melting curve analysis on the product of step (i).

12. The method is (1) A step of providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair and a detection probe for each target nucleic acid to be amplified, The universal primer contains a first universal sequence, and the first universal sequence is located at the 3' portion of the universal primer or constitutes the 3' portion of the universal primer. The target-specific primer pair can amplify the target nucleic acid, and includes a forward primer and a reverse primer. The forward primer includes a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid. The forward nucleotide sequence is located at the 3' end of the second universal sequence, and the forward nucleotide sequence is located at the 3' portion of the forward primer or constitutes the 3' portion of the forward primer. The reverse primer includes the first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid. The reverse nucleotide sequence is located at the 3' end of the first universal sequence, and the reverse nucleotide sequence is located at the 3' portion of the reverse primer or constitutes the 3' portion of the reverse primer. Under conditions enabling nucleic acid hybridization or annealing, the first universal sequence can hybridize or anneal to the complementary sequence of the second universal sequence. There is a difference between the second universal sequence and the first universal sequence, and the difference includes that one or more nucleotides located at the 3' end of the first universal sequence are independently deleted or substituted. The first universal sequence is not completely complementary to the complementary sequence of the forward primer. The detection probe includes a probe nucleotide sequence specific to the target nucleic acid, and is labeled with a reporter group and a quencher group. The reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group. A step in which the signal emitted when the detection probe hybridizes to its complementary sequence is different from the signal emitted when it is not hybridized to its complementary sequence. (2) Under conditions enabling nucleic acid amplification, amplifying the target nucleic acid in the sample via a PCR reaction using the universal primer and the target-specific primer pair. (3) performing melting curve analysis on the product of step (2) using the detection probe, and determining whether the target nucleic acid is present in the sample according to the result of the melting curve analysis; The method according to claim 11, comprising:

13. The method is as follows: (1) the universal primer is as defined in claim 4; (2) the target-specific primer pair is as defined in claim 5; (3) In step (2), mixing the sample with the universal primer, the target-specific primer pair, and the nucleic acid polymerase, performing a PCR reaction, and then, after the PCR reaction is completed, adding the detection probe to the product of step (2) and performing the melting curve analysis, or in step (2), mixing the sample with the universal primer, the target-specific primer pair and the detection probe, and the nucleic acid polymerase, performing a PCR reaction, and then, after the PCR reaction is completed, performing the melting curve analysis; (4) the detection probe comprises, or consists of, naturally occurring nucleotides, modified nucleotides, unnatural nucleotides, or any combination thereof; (5) the detection probe has a length of 15 nt to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, 90 nt to 100 nt, 100 nt to 200 nt, 200 nt to 300 nt, 300 nt to 400 nt, 400 nt to 500 nt, 500 nt to 600 nt, 600 nt to 700 nt, 700 nt to 800 nt, 800 nt to 900 nt, 900 nt to 1000 nt; (6) the detection probe has a 3'-OH terminus or the 3' terminus of the detection probe is blocked; (7) the detection probe is a self-quenching probe; (8) the reporter group of the detection probe is a fluorescent group, and the quencher group is a molecule or group capable of absorbing / quenching the fluorescence; (9) the detection probe has resistance to nuclease activity; (10) the detection probe is linear or has a hairpin structure; (11) each of the detection probes independently has the same or different reporter groups; In step (12), in order to obtain a curve of the signal intensity of each reporter group that varies according to the change in temperature, the product of step (2) is gradually heated or cooled, the signal emitted by the reporter group of each detection probe is monitored in real time, and then the curve is differentiated to obtain the melting curve of the product of step (2); and, In step (13), according to the melting peak (melting point) of the melting curve, the presence of the target nucleic acid corresponding to the melting peak (melting point) is determined; The method according to claim 12, having one or more technical features selected from:

14. Steps (1) to (3) of the method are the following steps (a) to (g): (a) A step of providing a sample containing one or more target nucleic acids, providing a universal primer, and providing a target-specific primer pair and a detection probe for each target nucleic acid to be amplified, wherein The step in which the universal primer, the target-specific primer pair, and the detection probe are as defined in claim 12; (b) A step of mixing the sample with the universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase; (c) A step of incubating the product of the previous step under conditions capable of nucleic acid denaturation; (d) A step of incubating the product of the previous step under conditions capable of nucleic acid annealing or hybridization; (e) A step of incubating the product of the previous step under conditions capable of nucleic acid extension; (f) A step of repeating or not repeating steps (c) to (e) one or more times; (g) A step of performing melting curve analysis on the product of the previous step; The method according to claim 12, which is performed by a protocol including:

15. The method is as follows: (1) The naturally occurring nucleotides are deoxyribonucleotides or ribonucleotides; (2) The unnatural nucleotides are peptide nucleic acids (PNA) or locked nucleic acids; (3) The 3'-end of the detection probe is blocked by adding a chemical moiety to the 3'-OH of the last nucleotide of the detection probe, by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide; (4) The 3'-end of the detection probe is blocked by adding biotin or alkyl to the 3'-OH of the last nucleotide of the detection probe; (5) The distance between the reporter group and the quencher group is 10 nt to 80 nt or more apart; (6) The reporter group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor (trademark) Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, Texas Red, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quencher group is selected from DABCYL, BHQ, Eclipse, and / or TAMRA; (8) The detection probe has resistance to 5'-nuclease activity; (9) The detection probe has resistance to 5'→3' exonuclease activity; (10) The detection probe has a backbone containing modifications for resistance to nuclease activity; (11) The detection probe has a backbone containing a modification selected from phosphorothioate ester bond, alkyl phosphotriester bond, aryl phosphotriester bond, alkyl phosphonate ester bond, aryl phosphonate ester bond, hydrogenated phosphate ester bond, alkyl phosphoramidate ester bond, aryl phosphoramidate ester bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification; (12) The detection probe is labeled with a reporter group at its 5'-end or upstream thereof, and labeled with a quencher group at its 3'-end or downstream thereof; and (13) The detection probe has the same reporter group, the product of step (2) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined according to the melting peak of the melting curve, or the detection probe has different reporter groups, the product of step (2) is subjected to melting curve analysis, and then the presence of the target nucleic acid is determined according to the signal type of the reporter group and the melting peak of the melting curve; The method according to claim 13, having one or more technical features selected from

16. A primer set comprising a universal primer and one or more target-specific primer pairs, wherein the universal primer comprises a first universal sequence, and the first universal sequence is located at the 3' portion of the universal primer or constitutes the 3' portion of the universal primer, each target-specific primer pair being capable of amplifying a target nucleic acid and comprising a forward primer and a reverse primer, wherein the forward primer comprises a second universal sequence and a forward nucleotide sequence specific to the target nucleic acid, the forward nucleotide sequence being located at the 3' end of the second universal sequence, and the forward nucleotide sequence being located at the 3' portion of the forward primer or constituting the 3' portion of the forward primer, wherein the reverse primer comprises the first universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, the reverse nucleotide sequence being located at the 3' end of the first universal sequence, and the reverse nucleotide sequence being located at the 3' portion of the reverse primer or constituting the 3' portion of the reverse primer, under conditions allowing hybridization or annealing of nucleic acids, the first universal sequence being capable of hybridizing or annealing to the complementary sequence of the second universal sequence, there being a difference between the second universal sequence and the first universal sequence, the difference including that one or more nucleotides located at the 3' end of the first universal sequence are independently deleted or substituted, and the first universal sequence not being completely complementary to the complementary sequence of the forward primer, a primer set.

17. The primer set is as follows: (1) the primer set comprises 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 50 or more target-specific primer pairs; (2) the universal primer is as defined in claim 4; and, (3) the target-specific primer pair is as defined in claim 5; The primer set according to claim 16, having one or more technical features selected from

18. A kit comprising the primer set according to claim 16 and one or more components selected from a nucleic acid polymerase, a reagent for nucleic acid amplification, a reagent for sequencing, a reagent for gene chip detection, a reagent for melting curve analysis, or any combination thereof.

19. The kit is as follows: (1) The nucleic acid polymerase is a template-dependent nucleic acid polymerase; (2) The reagent for nucleic acid amplification includes a working buffer for the enzyme, dNTPs, water, a solution containing ions, a single-stranded DNA binding protein, or any combination thereof; (3) The reagent for sequencing includes a working buffer for the enzyme, dNTPs, ddNTPs, water, a solution containing ions, a single-stranded DNA binding protein (SSB), a ligase, a nucleic acid linker, a sequencing primer, or any combination thereof; (4) The reagent for gene chip detection includes a working buffer for the enzyme, dNTPs, water, a hybridization buffer, a washing buffer, a labeling reagent, or any combination thereof; and (5) The reagent for melting curve analysis includes a detection probe, The kit according to claim 18, having one or more technical features selected from the above.

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