Detection method and device based on FRET effect

Abstract

The invention relates to the field of molecular biology, in particular to a detection method and device based on an FRET effect. According to the present invention, the linear relationship between the energy transfer efficiency (E) of the intramolecular FRET effect and the reciprocal R-1 of the linear distance between the intramolecular donor and the intramolecular receptor is firstly found, and the detection method based on the finding is provided, such that the mutation nucleic acid can be effectively detected so as to easily reduce the detection failure caused by the microbial mutation, the kit can realize early evolution early warning of viruses and real-time monitoring of virus variation, and can be widely applied to multiple fields of fluorescent mice, antibody preparation, molecular hybridization, genotyping, cancer mutation screening, clinical pathogen drug-resistant mutation detection and the like.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a detection method and apparatus based on the FRET effect. Background Technology

[0002] Quantitative Polymerase Chain Reaction (qPCR), a highly efficient and sensitive nucleic acid quantification technique, offers significant advantages such as real-time detection of the amplification process via fluorescence signals, dynamic tracking of product accumulation, no need for opening the container, and avoidance of aerosol contamination. It has been widely used for the rapid detection of infectious pathogens, providing laboratory diagnostic evidence for infectious diseases. Based on the fluorescence signal generation mechanism, qPCR technology can be divided into dye-based and probe-based methods. Dye-based methods lack target specificity and are susceptible to interference from primer dimers or non-specific amplification, thus limiting their application to broad-spectrum screening. In contrast, probe-based methods, based on the principle of fluorescence resonance energy transfer (FRET), design corresponding "reporter-quencher" dual-labeled nucleic acid probes, enabling precise binding to target sequences and ensuring high detection specificity. This makes them more suitable for detecting specific pathogens in complex clinical samples.

[0003] However, for TaqMan hydrolyzable probes, once a base mutation occurs in the target region, the probe becomes difficult to bind to, or after binding, it cannot be hydrolyzed by the 5'→3' exonuclease activity of Taq polymerase, thus rendering the target ineffective. In contrast, molecular beacon (MB) probes have a unique stem-loop structure and high signal-to-noise ratio, exhibiting high sensitivity and specificity for recognizing single-base mutation sequences. Therefore, they can tolerate viral mutations to some extent. However, this method can only detect known variants. When new variants emerge, MB probes must be redesigned, resulting in significant detection lag and difficulty in handling unknown variants. Furthermore, conventional MB systems are difficult to design and unstable; generally, MB probes become ineffective when the number of mutated bases in a variant is ≥2. These shortcomings cause existing qPCR technologies to face problems such as decreased sensitivity and increased false negative rates when dealing with emerging epidemics caused by highly variable viruses, leading to the rapid spread of variants and posing a significant threat to public health and epidemic prevention efforts. Therefore, developing new detection technologies for real-time monitoring of variants is urgently needed.

[0004] One of the theoretical cornerstones of traditional probe-based qPCR is the fluorescence resonance energy transfer (FRET) effect. This effect is a nonradiative energy transfer process that transfers energy from the excited state of a donor to the excited state of a receptor through dipole-dipole interactions between molecules. Förster was the first to elucidate its mechanism, so it is also called Förster resonance energy transfer. The basic principle of FRET is that when a fluorescent group (donor) is excited, its energy can be transferred nonradiatively to another nearby fluorescent group (receptor), resulting in a decrease in the fluorescence intensity of the donor. The receptor can emit a stronger characteristic fluorescence than itself, or it can not fluoresce (fluorescence quenching). This energy transfer phenomenon does not occur simply because there is an energy donor and an energy acceptor. Instead, it depends on the degree of spectral overlap, the distance between the donor and the acceptor, and their spatial orientation: (1) Spectral overlap: There is good overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (generally greater than 30%); (2) Distance dependence: When the distance between the donor and the acceptor ( R When the wavelength is less than 10 nm, the energy transfer efficiency of the FRET effect ( E ) and the distance between the donor and recipient ( R The sixth power of ) is inversely proportional ( E =1 / [1+( R / R 0) 6 ],in R 0 is the Förster radius, usually between 3 and 6 nm); (3) Relative orientation matching of the donor emission dipole moment and the acceptor absorption dipole moment: the FRET efficiency is higher when the donor and acceptor dipole moments are parallel than when they are perpendicular. The intermolecular interaction force between the donor and acceptor needs to be strong enough to make them an FRET pair. Among them, the interaction force between the donor and acceptor R -6 The distance dependence of the FRET effect makes it extremely sensitive to minute changes in distance. In traditional probe-based qPCR (such as Taqman hydrolyzed probes), this characteristic is transformed into a "fluorescence signal switch" mechanism: when the probe is intact, the energy of the 5' fluorescent reporter group is absorbed by the 3' fluorescent quencher group through the FRET effect, and the fluorescence signal is suppressed; during the annealing phase of the qPCR reaction, the probe and primers bind to the substrate simultaneously. If the Taqman probe is encountered during the primer extension synthesis of a new complementary DNA strand, the probe will be successfully cleaved and hydrolyzed under the action of the 5'→3' exonuclease activity of the thermostable DNA polymerase (Taq enzyme), and the fluorescent reporter group and the fluorescent quencher group will separate. When the distance between the fluorescent reporter group and the fluorescent quencher group exceeds 10 nm, the FRET effect terminates, the fluorescence signal is released, and the fluorescence signal is captured by the qPCR instrument.

[0005] qPCR, with its advantages of high sensitivity, high specificity, and early diagnosis, is widely used in various fields such as medicine, biological research, agriculture, food safety, and environmental science. Many studies based on qPCR technology focus on multiplex qPCR and high-throughput rapid detection. However, these studies have not paid attention to the underlying mechanism of intramolecular FRET effect, assuming that the energy transfer efficiency of FRET effect (…) E The sensitivity of qPCR is inversely proportional to the sixth power of the distance between the donor and recipient. Based on this logic, improvements and optimizations to qPCR technology are necessary. This is also a key reason why current qPCR technology experiences decreased sensitivity and increased false negative rates when dealing with emerging epidemics caused by highly variable viruses. For example, the design principle of TaqMan hydrolyzable probes allows them to specifically bind to the target sequence when detecting unmutated viruses and be hydrolyzed by Taq enzymes, thus separating the fluorescent reporter group and the fluorescent quencher group. At this point, the distance between the fluorescent reporter group and the fluorescent quencher group exceeds 10 nm, the FRET effect terminates, and a fluorescent signal is released. However, when detecting mutant strains, TaqMan hydrolyzable probes struggle to bind to the mutated target sequence, or after binding, they cannot be hydrolyzed by the 5'→3' exonuclease activity of Taq enzymes, keeping the probe intact. In this case, the distance between the fluorescent reporter group and the fluorescent quencher group is less than 10 nm, resulting in no fluorescent signal and thus detection failure.

[0006] The MB probe possesses a unique stem-loop structure due to the addition of a half-complementary base stem. A fluorescent reporter group and a fluorescent quencher group are labeled at the 5' and 3' ends of the two stems, respectively. When detecting unmutated viruses, the circular region of the MB probe can specifically bind to the target sequence, causing the complementary base stem region to open and form a linear molecule. This linear molecule is malleable, increasing the distance between the fluorescent reporter and quencher groups to greater than 10 nm, thereby releasing a fluorescent signal. Unlike TaqMan hydrolyzable probes, when detecting single-base mutants, the circular region of the MB probe can still mismatch with the target sequence. However, compared to complete pair binding, mismatch binding makes it difficult for the stem region to open and form a linear molecule, releasing a lower intensity fluorescent signal. This phenomenon is consistent with known... R -6 The distance-dependent FRET effect contradicts this, because if the known FRET effect were followed, the distance between the fluorescent reporter group and the fluorescent quencher group would be... R -6The distance dependence of the FRET effect makes it extremely sensitive to minute changes in distance. When the stem region of the MB probe is stretched but cannot form a substantially straight linear molecule, changes in the distance between the fluorescent reporter group and the fluorescent quencher group can only produce two results: no fluorescence signal is generated or a fluorescence signal with an intensity comparable to that when the MB probe is fully open is released. However, this anomaly has not attracted the attention of researchers, its underlying mechanism has not been thoroughly studied, and this has limited the widespread application of MB probes. Summary of the Invention

[0007] The first objective of this invention is to provide a detection method based on the FRET effect, comprising: obtaining the energy transfer efficiency E of the intramolecular FRET effect; determining the linear distance R between the intramolecular donor and acceptor according to the energy transfer efficiency E and a first functional relationship; wherein the linear distance R satisfies 6 nm < R < 13 nm; the variables in the first functional relationship are the energy transfer efficiency E and the linear distance R, and the first functional relationship is based on the energy transfer efficiency E and the reciprocal R of the linear distance between the intramolecular donor and acceptor. -1 The linear relationship was determined.

[0008] The present invention also provides a computer device, including a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the FRET-based detection method.

[0009] The present invention also provides a computer-readable storage medium having stored thereon computer program instructions, wherein the computer program instructions, when executed by a processor, implement the steps of the FRET-based detection method.

[0010] The present invention also provides a computer program product, including computer program instructions, wherein the computer program instructions, when executed by a processor, implement the steps of the FRET-based detection method.

[0011] This invention first discovered the energy transfer efficiency of the intramolecular FRET effect ( E ) is the reciprocal of the linear distance between the donor and acceptor within the molecule R -1 A linear relationship is presented, and a detection method based on this discovery is proposed, which can effectively detect mutated nucleic acids, help reduce detection failures caused by microbial mutations, and realize early warning of virus evolution and real-time monitoring of virus mutations. It can be widely used in fluorescent mice, antibody preparation, molecular hybridization, genotyping, cancer mutation screening, clinical pathogen resistance mutation detection and other fields. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of the intramolecular FRET effect of the probe of the present invention.

[0014] Figure 2 This represents the distance relationship of the FRET effect within the probe molecule in this embodiment of the invention.

[0015] Figure 3 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 5°C in the embodiments of the present invention.

[0016] Figure 4 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 15°C in the embodiments of the present invention.

[0017] Figure 5 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 25°C in this embodiment of the invention.

[0018] Figure 6 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 35°C in this embodiment of the invention.

[0019] Figure 7 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 45°C in this embodiment of the invention.

[0020] Figure 8 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 55°C in this embodiment of the invention.

[0021] Figure 9 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 60°C in the embodiments of the present invention.

[0022] Figure 10 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 65°C in this embodiment of the invention.

[0023] Figure 11 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 70°C in the embodiments of the present invention.

[0024] Figure 12 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 75°C in this embodiment of the invention.

[0025] Figure 13 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 85°C in this embodiment of the invention.

[0026] Figure 14 The relative fluorescence unit is the probe detection of the mutant sequence at an annealing temperature of 95°C in this embodiment of the invention.

[0027] Figure 15 The oligonucleotide sequence is shown in the embodiments of the present invention.

[0028] Figure 16 This demonstrates the detection capability of the 2D qPCR reaction system for Ectromelia virus mutant strains in this embodiment of the invention.

[0029] Figure 17 This demonstrates the detection capability of traditional probe-based qPCR reaction systems for Ectromelia virus mutants. Detailed Implementation

[0030] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0031] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B.

[0033] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0034] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0035] The numerical values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 1% can fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 75% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0036] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0037] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0038] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0039] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0040] In this invention, the "mutation" includes the substitution, insertion, or deletion of bases.

[0041] In practice, the functional relationships and threshold ranges mentioned in this invention can be confirmed based on the detection of known samples.

[0042] This invention discovers that when the fluorescent reporter group (donor) and the fluorescent quencher group (acceptor) are located within the same molecule (e.g., Figure 1 As shown (using the probe described below as an example), the FRET effect does not exhibit the R-value as previously understood. -6The distance dependence of the target nucleic acid is as follows: When the qPCR reaction is running, if the target nucleic acid is not present, the stem region of the probe will close rapidly within 1 second. At this time, the distance between the fluorescent reporter group and the fluorescent quencher group is less than 5 nm, and the fluorescent signal emitted by the fluorescent reporter group is absorbed by the fluorescent quencher group. When the target nucleic acid is present, the probe will hybridize with the target nucleic acid to become double-stranded. At this time, the rigidity of the circular sequence is greatly increased, and the complementary strand between the two stem regions will be opened. However, the actual distance between the fluorescent reporter group and the fluorescent quencher group is related to the degree of mutation of the target nucleic acid. If the target nucleic acid has not mutated, the distance between the fluorescent reporter group and the fluorescent quencher group will increase to more than 13 nm after the complementary strand is opened, and the fluorescent signal will be fully released. If the target nucleic acid has mutated, the fewer the number of mutated bases, the larger the distance between the fluorescent reporter group and the fluorescent quencher group after the complementary strand is opened. At this time, although the distance between the fluorescent reporter group and the fluorescent quencher group is less than 13 nm, some fluorescent signal will still be released. Only when the number of mutated bases exceeds a certain number, the complementary strand cannot be opened, and the distance is less than 5 nm, resulting in no fluorescent signal release.

[0043] This fact confirms that the FRET effect does not always exhibit R, as previously thought. -6 The distance dependency, because in R -6Under the distance-dependent relationship, when the distance between the fluorescent reporter group and the fluorescent quencher group is less than 10 nm, the fluorescence signal will initially be quenched as the distance gradually increases. When the distance increases to a certain critical value, the fluorescence signal will suddenly be fully released. Only when the distance between the fluorescent reporter group and the fluorescent quencher group is greater than 10 nm will the fluorescence signal be stably and fully released without being affected by the FRET effect. It is precisely because of this characteristic that traditional probes are almost unable to detect any fluorescence signal when facing mutant strains, resulting in false negative results. The intramolecular FRET effect phenomenon discovered in this invention breaks through the distance-dependent bottleneck of the traditional intermolecular FRET effect, confirming that the fluorescent reporter group and the fluorescent quencher group can release a certain amount of fluorescence within a distance range of greater than 6 nm and less than 13 nm. This gives it a strong adaptability when detecting mutant strains. When a mutant strain is detected, the qPCR instrument will capture a fluorescence signal with a lower intensity compared to the original strain (positive control). Therefore, traditional probe-based qPCR only uses the Ct value (the number of cycles required for the fluorescence signal to reach a set threshold) to interpret the two-dimensional coordinate system of the detection results, reflecting the presence or absence of amplified products and quantifying the target molecule. This invention, while retaining the Ct value for result interpretation, innovatively uses the second dimension of the two-dimensional coordinate system—relative fluorescence units (RFU)—to determine whether a mutant strain has been detected. This method is simple and rapid: if the relative fluorescence units are the same as the original strain (positive control), the original strain has been detected; if the relative fluorescence units are lower than the original strain (positive control), a mutant strain has been detected; if no fluorescence signal is detected, the result is negative.

[0044] Based on the above findings, this invention first provides a detection method based on the FRET effect, comprising: obtaining the energy transfer efficiency E of the intramolecular FRET effect; determining the linear distance R between the intramolecular donor and acceptor according to the energy transfer efficiency E and a first functional relationship; wherein the linear distance R satisfies 6 nm < R < 13 nm; the variables in the first functional relationship are the energy transfer efficiency E and the linear distance R, and the first functional relationship is based on the energy transfer efficiency E and the reciprocal R of the linear distance between the intramolecular donor and acceptor. -1 The linear relationship was determined.

[0045] In some specific implementations, the first functional relationship is as follows: E = k / R + C ,in k The slope C is the fluorescence constant.

[0046] In some embodiments, the detection method includes: obtaining the relative fluorescence unit F of the molecule; determining the linear distance R between the intramolecular donor and acceptor based on the relative fluorescence unit F and a second functional relationship; wherein the linear distance R satisfies 6 nm < R < 13 nm; the variables in the second functional relationship are the relative fluorescence unit F and the linear distance R, respectively, and the second functional relationship is based on the energy transfer efficiency E and the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship was determined.

[0047] In some embodiments, the detection method includes: acquiring the relative fluorescence unit F of a molecule; determining the internal conformation of the molecule based on the relative fluorescence unit F and a first threshold range; wherein, when the internal conformations of the molecule are different, the donor and acceptor within the molecule have different linear distances R; the linear distance R satisfies 6 nm < R < 13 nm; the first threshold range is determined based on the following method: according to the energy transfer efficiency E and the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship between the linear distance R and the internal conformation of the molecule was used to determine the threshold range of the relative fluorescence unit F associated with the internal conformation of the molecule.

[0048] In some embodiments, the detection method includes: obtaining the relative fluorescence unit F when the molecule binds to the analyte; determining the degree of matching of the analyte based on the relative fluorescence unit F and a second threshold range; wherein, when the degree of binding between the molecule and the analyte is different, the intramolecular donor and acceptor have different linear distances R; the linear distance R satisfies 6 nm < R < 13 nm; the second threshold range is determined based on the following method: based on the energy transfer efficiency E and the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship between the linear distance R and the degree of matching between the analyte and the analyte is used to determine the threshold range of the relative fluorescence unit F associated with the degree of matching between the analyte and the analyte.

[0049] In some embodiments, the molecule is an MB probe.

[0050] In some embodiments, the molecule is a probe (also referred to as a Qian Hybridization probe in this invention), and the analyte is the target nucleic acid. The probe, from its 5' end to its 3' end, sequentially comprises a fluorescent reporter group, a stem sequence, an alkyl spacer, a loop sequence, another alkyl spacer, a stem sequence, and a fluorescence quencher group. The two stem sequences are complementary, forming a hairpin structure. The loop sequence contains a region complementary to the target nucleic acid. The alkyl spacer is a hydrophobic straight-chain alkyl spacer. Using this probe further facilitates the detection of mutant nucleic acids that have a single base mutation with the target nucleic acid.

[0051] In some embodiments, the molecule is a probe, and the analyte is a nucleic acid to be tested; obtaining the relative fluorescence unit F when the molecule binds to the analyte specifically includes: obtaining the relative fluorescence unit of the fluorescence channel after each amplification cycle when the probe and the nucleic acid to be tested are subjected to qPCR reaction, and calculating the average relative fluorescence unit as the relative fluorescence unit F.

[0052] In some embodiments, the molecule is a probe, and the analyte is a nucleic acid to be tested; determining the matching degree of the analyte based on the relative fluorescence unit F and the second threshold range specifically includes: when the relative fluorescence unit F is higher than the background signal and lower than the average relative fluorescence unit measured when the molecule binds to the target nucleic acid, it is determined that the nucleic acid to be tested has at least one base mutation compared to the target nucleic acid.

[0053] In some implementations, the length of each stem region sequence is 7-9 bps. For example, the length of each stem region sequence can be 7 bps, 8 bps, or 9 bps. Setting stem region sequences of the above lengths will further facilitate the detection of mutant nucleic acids.

[0054] In some embodiments, the stem region sequence comprises any one of the following sets of sequences I) to III): I) 5'-TCAGGCG-3' and 5'-CGCCTGA-3'; II) 5'-CGAGCGGC-3' and 5'-GCCGCTCG-3'; III) 5'-CGAGCGGCT-3' and 5'-AGCCGCTCG-3'.

[0055] In some embodiments, the length of the loop region sequence is 23–43 bps. In some preferred embodiments, the length of the loop region sequence is 23–38 bps. Within the range of 23–38 bps, the longer the loop region sequence, the stronger the detection system's ability to identify mutant nucleic acids. When the loop region sequence is greater than 38 bps but less than or equal to 43 bps, the detection system's ability to identify mutant nucleic acids will decrease to some extent, but it can still detect mutant nucleic acids. By changing the length of the loop region sequence, the detection system can be tuned to meet different detection needs.

[0056] As an example, in some specific implementations, the length of the ring region sequence can be 23 bps, 24 bps, 25 bps, 26 bps, 27 bps, 28 bps, 29 bps, 30 bps, 31 bps, 32 bps, 33 bps, 34 bps, 35 bps, 36 bps, 37 bps, 38 bps, 39 bps, 40 bps, 41 bps, 42 bps, or 43 bps.

[0057] In some embodiments, the hydrophobic straight-chain alkyl spacer is a C3 spacer (hereinafter also referred to as spacerC3) or a C6 spacer.

[0058] In some specific embodiments, the fluorescent reporter group includes, but is not limited to, at least one of FAM (6-carboxyfluorescein), TET, TAMRA (tetramethylrhodamine), HEX (6-carboxy-4,7,10-triphenyl-2,5,8,11-tetraazaanthracene), ROX, JOE, Cy3, Cy5, Texas Red, the Alexa Fluor series (such as Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647), and the Quasar series (such as Quasar 570, Quasar 670, Quasar 705).

[0059] In some specific embodiments, the fluorescence quenching group includes, but is not limited to, at least one of the following: QSY series (such as QSY 7, QSY21, QSY 9, QSY 65), MBQ (Methyl-BBQ), DABCYL (4-(4'-((dimethylamino)phenyl)ethynyl)benzoic acid), TMRox, BHQ (Black Hole Quencher) series (such as BHQ-1, BHQ-2, BHQ-3), and Dark Quencher series (such as Dark Quencher 550, Dark Quencher 610).

[0060] In practical implementation, based on considerations of the overlap of absorption and emission spectra, compatibility of detection systems, and photostability, those skilled in the art can select and combine the fluorescent reporter groups and fluorescent quencher groups mentioned above or not mentioned, in accordance with common sense. This invention does not impose any particular limitations on them.

[0061] In some specific embodiments, the probe may also contain one or more modifications. Common modification methods include chemical modifications (such as thiolation, methylation, phosphorylation, locked nucleic acid (LNA) modification, peptide nucleic acid (PNA) modification, etc.) and functional modifications (such as biotin modification, digoxigenin (DIG) modification, quantum dot (QD) modification, etc.). Such technical solutions are also within the scope of protection of this invention.

[0062] In some specific implementations, multiple different probes (such as different loop sequences) can be used, thereby enabling the simultaneous detection of multiple target nucleic acids in multiplex amplification.

[0063] Those skilled in the art can combine the above-described embodiments with common sense to obtain more embodiments of the probe of the present invention.

[0064] In some embodiments, the nucleic acid to be tested has a mutation of 8 or fewer bases compared to the target nucleic acid.

[0065] As an example, in some specific embodiments, the nucleic acid to be tested has the following number of base mutations compared to the target nucleic acid: 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0066] In some implementations, the annealing temperature during the qPCR reaction of the probe with the nucleic acid to be tested is controlled to be 5°C to 70°C.

[0067] As an example, in some specific embodiments, the annealing temperature of the probe during the qPCR reaction with the nucleic acid to be tested can be controlled to be 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0068] In some specific embodiments, the target nucleic acid is derived from pathogenic microorganisms.

[0069] In some specific embodiments, the pathogenic microorganism includes one or more of bacteria and viruses.

[0070] In some specific embodiments, the nucleic acid to be tested described in this invention can be derived from a variety of clinical sample types, such as blood samples (whole blood, plasma, serum), body fluid samples (cerebrospinal fluid, urine, pleural and peritoneal fluid), tissue samples (biopsy tissue, surgically removed tissue), secretion and excretion samples (sputum, feces, vomit), and other samples (hair, nails, tissue fluid).

[0071] In some specific embodiments, the nucleic acid to be tested described in this invention can also be derived from various sample types such as food samples, environmental samples, and crop samples.

[0072] In some specific implementations, the nucleic acid to be tested or the target nucleic acid is derived from microorganisms, plants, or animals (such as mice).

[0073] In some specific implementations, the nucleic acid to be tested or the target nucleic acid is derived from humans.

[0074] In some embodiments, the reaction system of the qPCR reaction contains at least one of the following components: 16~62.5 ng / μL of subtilisin sodium, 50~100 mmol / L of Tris-HCl at pH 8.0±0.5, and 0.5× SYBR GreenI.

[0075] In some specific embodiments, the reaction system of the qPCR reaction contains 16 ng / μL, 20 ng / μL, 25 ng / μL, 29 ng / μL, 30 ng / μL, 31 ng / μL, 32 ng / μL, 33 ng / μL, 35 ng / μL, 40 ng / μL, 45 ng / μL, 50 ng / μL, 55 ng / μL, 60 ng / μL, or 62.5 ng / μL of sodium subtilisin.

[0076] In some specific embodiments, the reaction system of the qPCR reaction contains 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L or 100 mmol / L pH 8.0±0.5 (e.g. 7.5, 7.8, 8.0, 8.2 or 8.5).

[0077] In some embodiments, the qPCR reaction system contains 125-500 nmol / L (preferably 250 nmol / L) of probe.

[0078] In some specific embodiments, the qPCR reaction system contains probes at concentrations of 125 nmol / L, 150 nmol / L, 175 nmol / L, 200 nmol / L, 225 nmol / L, 240 nmol / L, 245 nmol / L, 250 nmol / L, 255 nmol / L, 260 nmol / L, 275 nmol / L, 300 nmol / L, 325 nmol / L, 350 nmol / L, 375 nmol / L, 400 nmol / L, 425 nmol / L, 450 nmol / L, 475 nmol / L, or 500 nmol / L.

[0079] In some embodiments, each 20 μL of the qPCR reaction system contains the following components: 1×RapidTaq Master Mix, 0.5×SYBR Green I, 50~100 mmol / L (preferably 100 mmol / L) Tris-HCl at pH 8.0±0.5, 2~4 vol% (preferably 3 vol%) DMSO, 16~62.5 ng / μL (preferably 31 ng / μL) sodium subtilisin, 125~500 nmol / L (preferably 250 nmol / L) probe, 250~500 nmol / L (preferably 500 nmol / L) forward primer, 250~500 nmol / L (preferably 500 nmol / L) reverse primer, and 1~2 μL (preferably 1 μL) DNA template, with the remainder being water.

[0080] In some specific embodiments, the conditions for the qPCR reaction are as follows: 1) 95 °C, 3 min; 1 cycle; 2) 95 °C, 5 s; annealing (temperature as described above), 1 s; 69~70 °C (preferably 70 °C), 1 s; 40 cycles.

[0081] The detection method of this invention can complete the detection within 1 hour, which is about 1 / 2 to 1 / 3 of the time of the traditional Taqman qPCR method, meeting the requirements of timeliness of detection results in large-scale detection scenarios.

[0082] All steps in the detection method of the present invention can be performed by a person, by a device, or some steps can be performed by a person and some steps can be performed by a device.

[0083] The detection method in this invention can be based on diagnostic purposes or non-diagnostic purposes. In some embodiments (such as when based on diagnostic purposes), some or all of the steps of the detection method (such as steps directly or indirectly related to the diagnostic result) can be implemented by a device.

[0084] In specific implementation, those skilled in the art can combine the above-described embodiments with common sense to obtain more embodiments of the detection method of the present invention.

[0085] The present invention also provides a computer device, including a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the FRET-based detection method.

[0086] The present invention also provides a computer-readable storage medium having stored thereon computer program instructions, wherein the computer program instructions, when executed by a processor, implement the steps of the FRET-based detection method.

[0087] The present invention also provides a computer program product, including computer program instructions, wherein the computer program instructions, when executed by a processor, implement the steps of the FRET-based detection method.

[0088] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0089] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0090] Example 1: Distance Relationship of Intramolecular FRET Effect in Qian Hybridization Probes As shown in Table 1, six detection lines for mousepox virus (MPV) were designed respectively. Ectromelia virusThe Qian Hybridization probes were designed with a stem region consisting of 7 fixed base pairs (5'-TCAGGCG-3' and 5'-CGCCTGA-3') and loop regions (probe-specific variations) of 38 bps, 33 bps, 28 bps, 23 bps, 18 bps, and 13 bps, respectively. All probes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. qPCR reactions were performed using different Qian Hybridization probes without the addition of Taq polymerase or substrate, with each reaction in triplicate. A BioRad C1000 Touch Thermal Cycler / CFX96TM REAL-TIME SYSTEM with 5 fluorescence channels was used for qPCR. The qPCR reaction system is shown in Table 2, and the qPCR reaction conditions are shown in Table 3. The relative fluorescence units of the Rox fluorescence channels were recorded after each amplification cycle, and the average relative fluorescence units were calculated. The results are shown in Table 3. Figure 2 .

[0091] Table 1. Information on mousepox virus Qian Hybridization probes

[0092] Table 2. qPCR reaction systems without Taq enzyme and substrate.

[0093] Table 3 qPCR reaction conditions

[0094] This invention cleverly utilizes variations in annealing temperature to control the opening and closing degree of the QianHybridization probe without adding Taq enzyme or substrate, thereby altering the distance between the fluorescent reporter group and the fluorescent quencher group. The intensity of the fluorescence signal released by the Qian Hybridization probe under different opening and closing degrees is monitored in real time, thus confirming the energy transfer efficiency of the intramolecular FRET effect. E The relationship between the distance between the donor and recipient is not a sixth-power dependent relationship. For example... Figure 2As shown, with the loop sequence length remaining constant, the detected relative fluorescence units did not change significantly as the annealing temperature increased from 5℃ to 35℃. At this point, the complementary strand of the stem region of the Qian Hybridization probe was closed, and the detected fluorescence signal was a background signal. When the annealing temperature increased to 45℃~75℃, the complementary strand of the stem region of the Qian Hybridization probe gradually opened under the influence of high temperature, and the distance between the fluorescent reporter group and the fluorescent quencher group gradually increased. Even if there was no substrate complementary to the probe in the reaction system, its relative fluorescence units still showed an increasing trend with the increase of annealing temperature. When the annealing temperature increased to above 85℃, the detected relative fluorescence units basically tended to stabilize. At this point, the stem region of the Qian Hybridization probe completely unchained, its conformation changed to a linear molecule, and the fluorescence signal was completely released. It can be seen that when the Qian Hybridization probe is in an "intermediate state" between being completely off and completely on, it still emits a fluorescent signal, and the fluorescence intensity increases linearly with the degree of probe opening. This fact contradicts the known FRET effect. If the known FRET effect were followed, the fluorescence signal between the fluorescent reporter group and the fluorescent quencher group would be significantly different. R -6 The distance dependence makes the FRET effect extremely sensitive to small changes in distance. As the probe is gradually turned on, the fluorescence signal will first be in a quenched state. When the distance between the fluorescent reporter group and the fluorescent quencher group increases to a certain critical value, the energy transfer efficiency will suddenly drop sharply, resulting in a sudden release of a large amount of fluorescence signal instead of showing a stable linear growth relationship.

[0095] Furthermore, compared to the annealing temperature of 5℃, when the annealing temperature reached 95℃, the relative fluorescence units of the V1KG-38 probe increased by 167.54%, the V1KG-33 probe by 202.17%, the V1KG-28 probe by 197.98%, the V1KG-23 probe by 158.03%, the V1KG-18 probe by 139.28%, and the V1KG-13 probe by 119.06%. It can be seen that, under the same increasing annealing temperature, different loop sequence lengths of the Qian Hybridization probes result in significant differences in the intensity of the emitted fluorescence signal. In summary, within the annealing temperature range of 5℃ to 95℃, when the annealing temperature remains constant, a longer loop sequence of the Qian Hybridization probe leads to a larger detected relative fluorescence unit. This change in relative fluorescence units is caused by the structure of the Qian Hybridization probe itself. Because the Qian Hybridization probe undergoes continuous and random Brownian motion during the qPCR reaction, the longer the loop sequence, the larger the "loop," and the more unstable the probe's spatial conformation becomes during Brownian motion. This leads to fluctuations in the FRET effect, resulting in more fluorescence signals. Furthermore, Brownian motion is temperature-dependent; the higher the temperature, the more vigorous the Brownian motion. This phenomenon is most pronounced after the annealing temperature increases to 45℃. Within the annealing temperature range of 45℃ to 95℃, the relative fluorescence units of the Qian Hybridization probe exhibit a typical stepwise increase with increasing loop sequence length: V1KG-38 > V1KG-33 > V1KG-28 > V1KG-18 > V1KG-13. This stepwise trend persists even after the Qian Hybridization probe's molecular conformation transforms into a linear molecule.

[0096] Example 2: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at 5°C annealing temperature As shown in Table 4, one ssDNA completely complementary to the Qian Hybridization probe (positive control) and four ssDNAs partially complementary to the Qian Hybridization probe (base mutation) were designed. The design of the Qian Hybridization probe was the same as in Example 1. All probes and ssDNAs were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Under conditions without Taq enzyme, different Qian Hybridization probes were mixed with the synthesized ssDNA for qPCR reactions, with ddH2O added as a negative control instead of ssDNA. Each reaction was performed in triplicate. A BioRad C1000 Touch Thermal Cycler / CFX96TM REAL-TIMESYSTEM with 5 fluorescence channels was used for qPCR. The qPCR reaction system is shown in Table 5, and the qPCR reaction conditions are shown in Table 6. The relative fluorescence units of the Rox fluorescence channels in the qPCR reaction were recorded after each amplification cycle, and the average relative fluorescence units were calculated. The calculation results are shown in Table 6. Figure 3 .

[0097] Table 4 ssDNA Sequences

[0098] Table 5. qPCR reaction system without Taq enzyme

[0099] Table 6 qPCR reaction conditions

[0100] Example 3: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 15℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 15°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 4 .

[0101] Example 4: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 25℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 25°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 5 .

[0102] Example 5: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 35℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 35°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 6 .

[0103] Example 6: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at 45℃ annealing temperature Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 45°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 7 .

[0104] Example 7: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 55℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 55°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 8 .

[0105] Example 8: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 60℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 60°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 9 .

[0106] Example 9: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 65℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 65°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 10 .

[0107] Example 10: Luminescence properties of the Qian Hybridization probe in detecting mutant sequences at annealing temperature of 70℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 70°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 11 .

[0108] Example 11: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 75℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 75°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 12 .

[0109] Example 12: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 85℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 85°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 13 .

[0110] Example 13: Luminescence properties of the Qian Hybridization probe when detecting mutant sequences at annealing temperature of 95℃ Without the addition of Taq polymerase, different Qian Hybridization probes were mixed with synthesized ssDNA for qPCR reactions. ddH2O was added instead of ssDNA as a negative control. Each reaction was performed in triplicate. The design of the Qian Hybridization probes was the same as in Example 1, and the design of the ssDNA was the same as in Example 2. Except for the annealing temperature being changed to 95°C, the qPCR instrument, qPCR reaction system, and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated. The calculation results are shown below. Figure 14 .

[0111] like Figures 3-14As shown, at annealing temperatures of 5℃ to 35℃, the QianHybridization probe is in a closed state if no ssDNA is present. When the loop region sequence length of the QianHybridization probe is 13 bps, even when ssDNA that is completely complementary to the loop region sequence is added to the reaction system, the detected relative fluorescence units are only about 3% higher than the negative control. This may be because the loop region sequence is too short, and the intermolecular forces formed by pairing and binding with the substrate are less than the binding forces within the same strand of the stem region, thus failing to open the stem region. On the other hand, the hydrogen bonds within the same strand of the stem region may have been successfully opened, but the distance between the two strands after opening is not significantly different from the distance between the original fluorescent reporter group and the fluorescent quencher group, so only a weak fluorescence signal is generated. Since the vertical distance between adjacent base pairs in a DNA molecule is 0.34 nm, the straight-line distance between the fluorescent reporter group and the fluorescent quencher group should be less than the vertical distance after the loop region sequence pairing and binding is "straightened out" (0.34 nm × 13 bps = 4.42 nm). Therefore, it can be inferred that when the QianHybridization probe is in a completely off state, the linear distance between the fluorescent reporter group and the fluorescent quencher group is actually less than 5 nm. When the loop sequence increases to 18–38 bps, specific hybridization with unmutated ssDNA will cause the stem region to open to varying degrees. The longer the loop sequence, the greater the degree of stem region opening, and the stronger the released fluorescence signal. Therefore, a linear distance between the fluorescent reporter group and the fluorescent quencher group of 6–13 nm (0.34 nm × 18 bps; 0.34 nm × 38 bps) is required to produce a significantly higher fluorescence signal than the background. Furthermore, as the number of ssDNA base mutations increases linearly to 1, 3, 5, and 7, the detected relative fluorescence units also show a similar linear decreasing trend. Even with 7-base mutations in ssDNA, a higher fluorescence intensity than the negative control can still be detected, indicating that the Qian Hybridization probe has high mutation tolerance.However, as the annealing temperature gradually increases, within the annealing temperature range of 45℃ to 75℃, when the Qian Hybridization probe is in a gradually opening "intermediate state," the influence of the loop region sequence length on the anti-mutation ability of the Qian Hybridization probe gradually increases. At an annealing temperature of 60℃, only Qian Hybridization probes with loop regions of 38 bps, 33 bps, and 28 bps can bind to ssDNA with 7 base mutations and produce fluorescence signals higher than the negative control. At an annealing temperature of 65℃, only Qian Hybridization probes with loop regions of 38 bps and 33 bps can bind to ssDNA with 7 base mutations and produce fluorescence signals higher than the negative control. At an annealing temperature of 70℃, only Qian Hybridization probes with loop regions of 38 bps can bind to ssDNA with 5 base mutations and produce fluorescence signals higher than the negative control. However, at an annealing temperature of 75℃, all Qian Hybridization probes... The hybridization probes failed to bind to mutated ssDNA and produce fluorescence signals higher than the negative control. This indicates that as the annealing temperature increases, the rigid stress formed by the binding of the Qian Hybridization probe loop sequence to ssDNA, combined with the high temperature, acts on the stem region. The higher the temperature, the greater the degree of stem region opening. If the loop sequence is shorter and the number of mutated ssDNA bases is greater, the rigid stress effect during mismatch hybridization is less than the degree of stem region opening caused by high temperature, resulting in a fluorescence intensity higher than the background signal and detection failure. When the annealing temperature rises above 85℃, the Qian Hybridization probe stem region is fully open. Even with a loop length of 38 bps and a perfect match with the unmutated ssDNA, a fluorescence intensity higher than the background signal cannot be produced. This indicates that the linear distance between the fluorescent reporter group and the fluorescent quencher group exceeds the range of the FRET effect. Therefore, it can be inferred that when the Qian Hybridization probe is fully open, the linear distance between the fluorescent reporter group and the fluorescent quencher group should be greater than 13 nm (0.34 nm × 38 bps). Therefore, the present invention is based on. Figures 3-14 The fluorescence intensity at a specific annealing temperature shows a linear decrease with increasing substrate mutation number, leading to the derivation of the relationship between the donor and acceptor distances ( ). R When the wavelength is greater than 6 nm and less than 13 nm, the energy transfer efficiency of the intramolecular FRET effect ( E )formula:

[0112] in, k The slopeR The distance between the fluorescent reporter group and the fluorescent quencher group should be greater than 6 nm and less than 13 nm. C The fluorescence constant is given. The formula derived in this invention proves that within the same molecule, when the distance (R) between the donor and acceptor is greater than 6 nm and less than 13 nm, the energy transfer efficiency of the FRET effect (…) E The actual energy transfer efficiency is inversely proportional to the first power of the distance between the donor and acceptor. E ) and relative fluorescence units ( F The distance between the fluorescent reporter group and the fluorescent quencher group is inversely proportional to the distance between them. R The number of bases (N) in the loop region sequence of the Qian Hybridization probe is directly proportional to the number of bases in the target sequence. Therefore, when the target sequence mutates, a "vacuole" region due to base mismatch will be generated in the pairing region where the Qian Hybridization probe hybridizes. The presence of this "vacuole" causes conformational distortion of the probe. The more mutated bases in the target sequence, the larger the "vacuole," the greater the degree of conformational distortion of the probe, and the smaller the distance between the fluorescent reporter group and the fluorescent quencher group. R -1 Under distance-dependent conditions, fluorescence intensity exhibits a linear decreasing trend.

[0113] Example 14: 2D qPCR reaction system for mousepox virus ( Ectromelia virus Detection capability of mutant strains like Figure 15 As shown, a special oligonucleotide sequence was designed containing 6 mousepox viruses that are either completely complementary (positive control) or partially complementary (base mutation) to the Qian Hybridization probe. Ectromelia virus Sequence fragments (V1-0, V1-1, V1-3, V1-5, V1-7, V1-1-7), with uppercase letters indicating the mutated bases. Two additional QianHybridization probes were added to the design based on Example 1 (Table 7). According to Tables 8 and 9, different QianHybridization probes, detection primer pairs, and oligonucleotides were mixed for qPCR reactions. The qPCR reaction conditions are shown in Table 10, and the qPCR results are as follows. Figure 16 All probes, oligonucleotides, and primers were designed and then synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0114] Table 7. Detection of mousepox virus in 2D qPCR reaction system ( Ectromelia virus QianHybridization probe

[0115] Table 8 Mousepox virus ( Ectromelia virus 2D qPCR reaction system for sequence fragments

[0116] Table 9 Mousepox virus ( Ectromelia virus Detection primer pairs for sequence fragments

[0117] Table 10 Mousepox virus ( Ectromelia virus 2D qPCR reaction conditions for sequence fragments

[0118] like Figure 16 As shown, the 2D qPCR detection results of mousepox virus sequence fragments are reflected by amplification curves in a two-dimensional matrix coordinate system. This invention, while retaining the traditional qPCR method's interpretation of the Ct value of the amplification curve, innovatively uses the second dimension of the two-dimensional matrix coordinate system—the relative fluorescence units (RFU) of the amplification curve—to determine whether a mutant strain has been generated. This method is simple, intuitive, and rapid. It can be seen that this invention has high sensitivity in detecting mutant strains, even when the oligonucleotide substrate concentration is diluted to 10... -6At this time, a strong fluorescence signal can still be detected, and the detection ability is positively correlated with the length of the Qian Hybridization probe loop sequence. When the Qian Hybridization probe loop sequence length is 18 bps, the present invention has difficulty identifying mutant strains and can only detect fluorescence signals of the positive control (V1-0). As the loop sequence length continues to increase, the present invention begins to show its ability to identify mutant strains, and can detect fluorescence signals of up to 8 base mutation sequence fragments. When the loop sequence length increases to 23-38 bps, compared with the positive control, the Ct value of the mutant strain changes less, but the detected relative fluorescence units change significantly. As the number of base mutations increases, the detected relative fluorescence units gradually decrease, which can be used to qualitatively determine the degree of mutation in the mutant strain. However, when the loop sequence length increases to 43 bps, the sensitivity of the present invention to the number of base mutations begins to decrease. Compared with the positive control, the detected relative fluorescence units only begin to show a significant decrease when the number of base mutations is greater than 3. When the length of the loop region sequence increases to 48 bps, the fluorescence signals produced by almost all mutant strains are similar to those of the positive control, making it difficult to accurately determine whether a mutant strain has been produced by changes in fluorescence intensity. However, on the other hand, the anti-mutation ability of the detection system of this invention reaches its peak at this time. The number of mutant bases has little effect on the linear distance between the fluorescent reporter group and the fluorescent quencher group of the Qian Hybridization probe. Therefore, when a mutant strain appears, there will be no false negative results, which can effectively eliminate the risk of missed detection.

[0119] Comparative Example 1: Traditional probe-based qPCR reaction system against mousepox virus ( Ectromelia virus Detection capability of mutant strains Based on the loop region sequence of the V1KG-23 Qian Hybridization probe, a TaqMan hydrolyzable probe was synthesized. The qPCR reaction system was prepared and the qPCR reaction was performed according to the instructions of the commercial probe assay kit. The source and amount of oligonucleotides and detection primers were the same as in Example 14. The qPCR results are as follows: Figure 17 .

[0120] like Figure 17As shown, compared to the present invention, the traditional Taqman hydrolytic probe cannot identify any mutant strains, and only a sufficiently strong fluorescent signal was detected in the positive control (V1-0). When the oligonucleotide sequence fragment undergoes a mutation of 1 to 8 bases, no fluorescent signal is detected. This is because the Taqman hydrolytic probe can only specifically bind to the unmutated target sequence. After binding, it is hydrolyzed by the 5'→3' exonuclease activity of Taq enzyme, thereby separating the fluorescent reporter group and the fluorescent quencher group to a distance of more than 10 nm, terminating the FRET effect and releasing a fluorescent signal. However, when faced with mutant strains, the Taqman hydrolytic probe will have difficulty binding to the mutated target sequence, or after binding, it will not be hydrolyzed by the 5'→3' exonuclease activity of Taq enzyme, keeping the probe intact. In this case, the distance between the fluorescent reporter group and the fluorescent quencher group is less than 10 nm, so no fluorescent signal is generated, resulting in detection failure.

[0121] The above results fully demonstrate that the 2D qPCR system of this invention, through a breakthrough in the FRET effect theory, reveals the energy transfer law of the intramolecular FRET effect, and solves the dilemma of mutation escape and detection failure in the traditional qPCR method. It not only has extremely strong anti-mutation ability, but can also provide early warning of viral evolution through changes in fluorescence intensity in the 2D matrix coordinate system, so as to deal with the off-target risk when detecting highly variable pathogens. It is of great significance for real-time monitoring of pathogen mutation and formulation of prevention and control strategies during the epidemic.

[0122] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A detection method based on the FRET effect, comprising: Obtain the energy transfer efficiency E of the intramolecular FRET effect; Based on the energy transfer efficiency E and the first functional relationship, the linear distance R between the intramolecular donor and acceptor is determined; Among them, the straight-line distance R satisfies 6 nm < R < 13 nm; The variables in the first functional relationship are energy transfer efficiency E and linear distance R, respectively. The first functional relationship is based on the reciprocal of the energy transfer efficiency E and the linear distance R between the intramolecular donor and acceptor. -1 The linear relationship was determined.

2. The detection method based on the FRET effect according to claim 1, comprising: Obtain the relative fluorescence unit F of the molecule; Based on the relative fluorescence unit F and the second function relationship, the linear distance R between the intramolecular donor and acceptor is determined; Among them, the straight-line distance R satisfies 6 nm < R < 13 nm; The variables in the second functional relationship are the relative fluorescence unit F and the linear distance R, respectively. The second functional relationship is based on the energy transfer efficiency E and the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship was determined.

3. The detection method based on the FRET effect according to claim 1, comprising: Obtain the relative fluorescence unit F of the molecule; The internal conformation of the molecule is determined based on the relative fluorescence unit F and the first threshold range; Wherein, when the internal conformation of the molecule is different, the donor and acceptor within the molecule have different linear distances R; the linear distance R satisfies 6 nm < R < 13 nm; The first threshold range is determined based on the following method: the energy transfer efficiency E is divided by the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship between the linear distance R and the internal conformation of the molecule was used to determine the threshold range of the relative fluorescence unit F associated with the internal conformation of the molecule.

4. The detection method based on the FRET effect according to claim 1, comprising: Obtain the relative fluorescence unit F when the molecule binds to the analyte; The degree of matching of the analyte is determined based on the relative fluorescence unit F and the second threshold range; Wherein, when the degree of binding between the molecule and the analyte is different, the intramolecular donor and acceptor have different linear distances R; the linear distance R satisfies 6 nm < R < 13 nm; The second threshold range is determined based on the following method: the energy transfer efficiency E is divided by the reciprocal of the relative fluorescence unit F. -1 The reciprocal of the straight-line distance R -1 The linear relationship between the linear distance R and the degree of matching between the analyte and the analyte is used to determine the threshold range of the relative fluorescence unit F associated with the degree of matching between the analyte and the analyte.

5. The detection method based on the FRET effect according to any one of claims 1 to 4, wherein, The molecule is a probe, and the substance to be tested is the nucleic acid to be tested; The probe comprises, from the 5' end to the 3' end, a fluorescent reporter group, a stem region sequence, an alkyl spacer, a ring region sequence, an alkyl spacer, a stem region sequence, and a fluorescent quencher group, in sequence. Among them, the two stem region sequences can be complementary and paired, so that the probe forms a hairpin structure; The loop sequence contains regions that are complementary to the target nucleic acid; The alkyl spacer is a hydrophobic straight-chain alkyl spacer.

6. The detection method based on the FRET effect according to claim 4, wherein, The molecule is a probe, and the substance to be tested is the nucleic acid to be tested; The specific components of the relative fluorescence unit F when the molecule binds to the analyte include: Obtain the relative fluorescence units of the fluorescence channel after each amplification cycle when the probe and the nucleic acid to be tested are used in a qPCR reaction, and calculate the average relative fluorescence units as the relative fluorescence unit F.

7. The detection method based on the FRET effect according to claim 4, wherein, The molecule is a probe, and the substance to be tested is the nucleic acid to be tested; The determination of the matching degree of the analyte based on the relative fluorescence unit F and the second threshold range specifically includes: When the relative fluorescence unit F is higher than the background signal but lower than the average relative fluorescence unit measured when the molecule binds to the target nucleic acid, it is determined that the nucleic acid to be tested has at least one base mutation compared to the target nucleic acid.

8. The detection method based on the FRET effect according to claim 5, wherein, Each of the stem region sequences is 7-9 bps in length.

9. The detection method based on the FRET effect according to claim 5, wherein, The stem region sequence comprises any one of the following sets of sequences (I) to (III): I) 5'-TCAGGCG-3' and 5'-CGCCTGA-3'; II) 5'-CGAGCGGC-3' and 5'-GCCGCTCG-3'; III) 5'-CGAGCGGCT-3' and 5'-AGCCGCTCG-3'.

10. The detection method based on the FRET effect according to claim 5, wherein, The length of the ring region sequence is 23~43 bps.

11. The detection method based on the FRET effect according to claim 5, wherein, The length of the ring region sequence is 23~38 bps.

12. The detection method based on the FRET effect according to claim 5, wherein, The hydrophobic straight-chain alkyl spacer is a C3 spacer or a C6 spacer.

13. The detection method based on the FRET effect according to claim 5, wherein, The nucleic acid to be tested has a mutation of less than 8 bases compared to the target nucleic acid.

14. The detection method based on the FRET effect according to claim 5, wherein, The annealing temperature for the probe and the nucleic acid to be tested in the qPCR reaction is controlled to be 5℃~70℃.

15. A computer apparatus comprising a memory, a processor, and a computer program stored in the memory, wherein, The processor executes the computer program to implement the steps of the detection method based on the FRET effect according to any one of claims 1 to 14.

16. A computer-readable storage medium having stored thereon computer program instructions, wherein, When the computer program instructions are executed by the processor, they implement the steps of the detection method based on the FRET effect as described in any one of claims 1 to 14.

17. A computer program product comprising computer program instructions, wherein, When the computer program instructions are executed by the processor, they implement the steps of the detection method based on the FRET effect as described in any one of claims 1 to 14.

Images