A detection method and device based on FRET effect

Abstract

The present application relates to the field of molecular biology, in particular, to a detection method and device based on FRET effect. The present application first discovers that the energy transfer efficiency of intramolecular FRET effect E ) and the reciprocal of the linear distance of intramolecular donor and acceptor R ‑1 present a linear relationship, and proposes a detection method based on the discovery, which can effectively detect mutant nucleic acids, is beneficial to reduce the detection failure caused by microbial mutation, can realize early evolution warning and real-time monitoring of virus variation, and can be widely applied to multiple fields such as fluorescent mouse, antibody preparation, molecular hybridization, genotyping, cancer mutation screening, clinical pathogen drug resistance mutation detection and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular, to a detection method and device based on FRET effect. BACKGROUND

[0002] Quantitative Polymerase Chain Reaction (qPCR) as a highly efficient and sensitive nucleic acid quantification technology, has been widely used in the rapid detection of infectious pathogenic microorganisms, and provides laboratory diagnostic basis for the diagnosis of infectious diseases, because it can detect the amplification process in real time through fluorescence signal, dynamically track product accumulation, avoid aerosol pollution without opening cover operation, and has more significant advantages. According to the generation mechanism of fluorescence signal, qPCR technology can be divided into dye method and probe method. The dye method lacks target specificity and is easily interfered by primer dimer or non-specific amplification, so it is only suitable for broad-spectrum screening and has great application limitations. In contrast, the probe method is based on the principle of Fluorescence Resonance Energy Transfer (FRET) to design corresponding "reporter-quencher" double-labeled nucleic acid probes, so as to accurately bind to the target sequence, ensuring high specificity of detection, and is more suitable for the detection of specific pathogens in complex clinical samples.

[0003] However, for Taqman hydrolysis probe, once the target region it detects has a base mutation, the probe will be difficult to bind to it, or it will not be hydrolyzed by the 5'→3' exonuclease activity of Taq enzyme after binding, thereby causing the target to fail. In contrast, the Molecular Beacon (MB) probe has a unique stem-loop structure and high signal-to-noise ratio characteristics, and has high sensitivity and specificity for recognizing single-base mutant sequences, so it can tolerate virus mutations to some extent, but this method can only detect known mutant strains, and when new mutant strains appear, the MB probe still needs to be redesigned, and the detection not only has serious lag, but also is difficult to cope with unknown mutant strains. In addition, the probe design of the conventional MB system is difficult, the system is unstable, and generally when the number of mutant bases of the mutant strain is ≥2, the MB probe will also fail. These defects make the existing qPCR technology face problems such as decreased sensitivity and rising false negative rate when dealing with new epidemics caused by highly variable viruses, thereby causing the mutant strain to spread rapidly in a short period of time, bringing great hidden dangers to the safety of the population and epidemic prevention work. Therefore, it is urgent to develop new detection technology to realize real-time monitoring of mutant strains.

[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-dependent relationship of FRET effect makes FRET effect extremely sensitive to the slight change of distance, and when the stem region of MB probe is pulled apart but cannot form a substantially straight linear molecule, the change of distance between the fluorescence reporter group and the fluorescence quencher group can only produce two results: no fluorescence signal or release a fluorescence signal comparable to the intensity when the MB probe is fully open. However, this abnormal phenomenon has not attracted the attention of researchers, and the internal mechanism has not been studied in depth, which also limits the popularization and application of MB probe. SUMMARY

[0007] The first object of the present application is to provide a detection method based on FRET effect, which comprises: obtaining the energy transfer efficiency E of intramolecular FRET effect; determining the linear distance R of 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 respectively, and the first functional relationship is determined according to the linear relationship between the energy transfer efficiency E and the reciprocal of the linear distance R of intramolecular donor and acceptor. -1

[0008] The present application also provides a computer device comprising a memory, a processor and a computer program stored on the memory, wherein the processor executes the computer program to realize the steps of the detection method based on FRET effect.

[0009] The present application also provides a computer readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to realize the steps of the detection method based on FRET effect.

[0010] The present application also provides a computer program product comprising computer program instructions, wherein the computer program instructions are executed by a processor to realize the steps of the detection method based on FRET effect.

[0011] The present application first discovers that the energy transfer efficiency of intramolecular FRET effect ( E ) and the reciprocal of the linear distance of intramolecular donor and acceptor R -1 present a linear relationship, and proposes a detection method based on this discovery, which can effectively detect mutant nucleic acids, is beneficial to reduce the detection failure caused by microbial mutation, can realize early warning of virus evolution and real-time monitoring of virus variation, and can be widely used in many fields such as fluorescent mice, antibody preparation, molecular hybridization, genotyping, cancer mutation screening, clinical pathogen drug resistance mutation detection and the like. BRIEF DESCRIPTION OF DRAWINGS

[0012] ​In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings required to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figure 1 Schematic diagram of the intramolecular FRET effect of the probe of the present application.

[0014] Figure 2 Distance relationship of the intramolecular FRET effect of the probe in the embodiment of the present application.

[0015] Figure 3 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 5°C in the embodiment of the present application.

[0016] Figure 4 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 15°C in the embodiment of the present application.

[0017] Figure 5 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 25°C in the embodiment of the present application.

[0018] Figure 6 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 35°C in the embodiment of the present application.

[0019] Figure 7 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 45°C in the embodiment of the present application.

[0020] Figure 8 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 55°C in the embodiment of the present application.

[0021] Figure 9 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 60°C in the embodiment of the present application.

[0022] Figure 10 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 65°C in the embodiment of the present application.

[0023] Figure 11 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 70°C in the embodiment of the present application.

[0024] Figure 12 Relative fluorescence unit when the probe detects the mutant sequence at an annealing temperature of 75°C in the embodiment of the present application.

[0025] Figure 13 The relative fluorescence unit when the probe detects the mutant sequence at the annealing temperature of 85°C in the embodiment of the present application.

[0026] Figure 14 The relative fluorescence unit when the probe detects the mutant sequence at the annealing temperature of 95°C in the embodiment of the present application.

[0027] Figure 15 The oligonucleotide sequence in the embodiment of the present application.

[0028] Figure 16 The detection ability of the 2D qPCR reaction system in the embodiment of the present application to the mutant strain of ectromelia virus.

[0029] Figure 17 The detection ability of the traditional probe qPCR reaction system to the mutant strain of ectromelia virus. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and explanatory of the present application and are not intended to limit the present application, and those skilled in the art can make various modifications and changes to the present application without departing from the scope or spirit of the present application. For example, the features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0031] Unless otherwise defined, all terms (including technical and scientific terms) used in the disclosure of the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. By further guidance, the subsequent definitions are used to better understand the teachings of the present application. The terms used in the specification of the present application herein are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0032] The selection range of the terms "and / or", "or / and", and "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or 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 the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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. -6When the qPCR reaction is running, if there is no target nucleic acid, the stem region of the probe will quickly close within 1 s, at which 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 there is a target nucleic acid, the probe will hybridize with the target nucleic acid to become double-stranded, at which time the stiffness of the loop region sequence is greatly increased, and the complementary strands between the two stem region sequences are 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 does not mutate, the opening of the complementary strands will increase the distance between the fluorescent reporter group and the fluorescent quencher group to more than 13 nm, at which time the fluorescent signal is completely released. If the target nucleic acid mutates, the fewer the number of mutated bases, the greater the distance between the fluorescent reporter group and the fluorescent quencher group after the complementary strands are pulled apart. At this time, although the distance between the fluorescent reporter group and the fluorescent quencher group is less than 13 nm, there will still be some fluorescent signal released. Only when the number of mutated bases exceeds a certain number, the complementary strands cannot be opened, and the distance is less than 5 nm, resulting in no fluorescent signal release.

[0043] This fact proves that the FRET effect does not always present the R -6 distance-dependent relationship as previously recognized, because in the R -6The distance-dependent relationship is that when the distance between the fluorescence reporter group and the fluorescence quenching group is less than 10 nm, the fluorescence signal is quenched as the distance gradually increases, when the distance increases to a certain critical value, the fluorescence signal is suddenly released, and only when the distance between the fluorescence reporter group and the fluorescence quenching group is greater than 10 nm, the fluorescence signal is stably released without the influence of the FRET effect. The traditional probe is in such characteristics that when the mutant strain is detected, the qPCR instrument can hardly detect any fluorescence signal, and the negative result is misjudged. The intramolecular FRET effect phenomenon discovered by the present application breaks the distance-dependent bottleneck of the traditional intermolecular FRET effect, and it is proved that the fluorescence reporter group and the fluorescence quenching group can also release a certain amount of fluorescence in the distance range of greater than 6 nm and less than 13 nm, which gives it strong adaptability when detecting mutant strains. When the mutant strain is detected, the qPCR instrument will capture a fluorescence signal with lower intensity compared with the original strain (positive control). Therefore, the traditional probe method qPCR always has only one dimension of Ct value (the number of cycles experienced when the fluorescence signal reaches the set threshold) in the two-dimensional coordinate system to reflect the presence or absence of the amplification product, and to quantify the target molecule. On the basis of retaining the Ct value for result judgment, the present application directly reads the second dimension data of the two-dimensional coordinate system, the relative fluorescence unit (RFU), to judge whether the mutant strain is detected, which is simple and fast. If the relative fluorescence unit is the same as the original strain (positive control), it means that the original strain is detected; if the relative fluorescence unit is lower than the original strain (positive control), it means that the mutant strain is detected; and if no fluorescence signal is detected, it is judged as a negative result.

[0044] Based on the above finding, the present application first provides a detection method based on FRET effect, which comprises: obtaining the energy transfer efficiency E of the intramolecular FRET effect; determining the straight-line distance R of the intramolecular donor and acceptor according to the energy transfer efficiency E and a first functional relationship; wherein the straight-line distance R satisfies 6 nm < R < 13 nm; the variable in the first functional relationship is the energy transfer efficiency E and the reciprocal of the straight-line distance R of the intramolecular donor and acceptor respectively, and the first functional relationship is determined according to the linear relationship between the energy transfer efficiency E and the reciprocal of the straight-line distance R of the intramolecular donor and acceptor. -1

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

[0046] ​In some embodiments, the detection method comprises: obtaining a relative fluorescence unit F of the molecule; determining a straight-line distance R between the donor and the acceptor in the molecule according to the relative fluorescence unit F and a second functional relationship, wherein the straight-line distance R satisfies 6 nm < R < 13 nm; and the variable in the second functional relationship is the relative fluorescence unit F and the straight-line distance R respectively, and the second functional relationship is determined according to a linear relationship between an energy transfer efficiency E and an inverse of the relative fluorescence unit F -1 and an inverse of the straight-line distance R -1 respectively.

[0047] In some embodiments, the detection method comprises: obtaining a relative fluorescence unit F of the molecule; determining an internal conformation of the molecule according to the relative fluorescence unit F and a first threshold range, wherein the donor and the acceptor in the molecule have different straight-line distances R when the internal conformation of the molecule is different; the straight-line distance R satisfies 6 nm < R < 13 nm; and the first threshold range is determined according to a linear relationship between an energy transfer efficiency E and an inverse of the relative fluorescence unit F -1 and an inverse of the straight-line distance R -1 respectively, and a correlation between the straight-line distance R and the internal conformation of the molecule.

[0048] In some embodiments, the detection method comprises: obtaining a relative fluorescence unit F of the molecule when the molecule is combined with a to-be-detected substance; determining a matching degree of the to-be-detected substance according to the relative fluorescence unit F and a second threshold range, wherein the donor and the acceptor in the molecule have different straight-line distances R when the combination degree of the molecule and the to-be-detected substance is different; the straight-line distance R satisfies 6 nm < R < 13 nm; and the second threshold range is determined according to a linear relationship between an energy transfer efficiency E and an inverse of the relative fluorescence unit F -1 and an inverse of the straight-line distance R -1 respectively, and a correlation between the straight-line distance R and the matching degree of the to-be-detected substance.

[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 herein), and the substance to be detected is a nucleic acid to be detected; the probe comprises, from 5' end to 3' end, a fluorescent reporter group, a stem region sequence, an alkyl spacer, a loop region sequence, an alkyl spacer, a stem region sequence, and a fluorescent quencher group in sequence; wherein the two stem region sequences can pair with each other to form a hairpin structure of the probe; the loop region sequence comprises a region that can pair with the target nucleic acid; and the alkyl spacer is a hydrophobic straight-chain alkyl spacer. By using the above probe, a mutant nucleic acid having a one-base mutation with respect to the target nucleic acid can be further detected.

[0051] In some embodiments, the molecule is a probe, and the substance to be detected is a nucleic acid to be detected; and the relative fluorescence unit F of the molecule when combined with the substance to be detected is specifically obtained by: obtaining the relative fluorescence unit of the probe after each amplification cycle in a qPCR reaction with the nucleic acid to be detected, and calculating the average relative fluorescence unit as the relative fluorescence unit F.

[0052] In some embodiments, the molecule is a probe, and the substance to be detected is a nucleic acid to be detected; and the matching degree of the substance to be detected is determined according to the relative fluorescence unit F and the second threshold range, specifically by: when the relative fluorescence unit F is higher than the background signal and lower than the average relative fluorescence unit measured when the molecule is combined with the target nucleic acid, it is determined that the nucleic acid to be detected has at least one base mutation compared with the target nucleic acid.

[0053] In some embodiments, each of the stem region sequences has a length of 7-9 bps. For example, each of the stem region sequences can have a length of 7 bps, 8 bps, or 9 bps. By setting the stem region sequence to have the above length, the detection of the mutant nucleic acid can be further facilitated.

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

[0055] In some embodiments, the loop region sequence has a length of 23-43 bps. In some preferred embodiments, the loop region sequence has a length of 23-38 bps. Within the range of 23-38 bps, the longer the loop region sequence, the stronger the discrimination ability of the detection system for the mutant nucleic acid. When the length of the loop region sequence is greater than 38 bps and less than or equal to 43 bps, the discrimination ability of the detection system for the mutant nucleic acid will decrease to a certain extent, but the detection of the mutant nucleic acid can still be achieved. By changing the length of the loop region sequence, the detection system can be regulated to meet different detection needs.

[0056] By way of example, in some specific embodiments, the loop region sequence can have a length of 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 linear 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-tetraazatetra- xanthene), ROX, JOE, Cy3, Cy5, Texas Red, Alexa Fluor series (such as Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647), Quasar series (such as Quasar 570, Quasar 670, Quasar 705).

[0059] In some specific embodiments, the fluorescent quencher group includes, but is not limited to, at least one of QSY series (such as QSY 7, QSY 21, 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), Dark Quencher series (such as Dark Quencher 550, Dark Quencher 610).

[0060] In specific implementations, based on the consideration of the overlap of absorption and emission spectra, detection system compatibility and optical stability, one skilled in the art can combine the above-mentioned or unmentioned fluorescent reporter groups and fluorescent quencher groups with common sense, and the present application does not make special limitations thereto.

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

[0062] In some specific embodiments, multiple different (such as different loop region sequences) probes can be used, so that multiple target nucleic acids can be detected simultaneously in multiplex amplification.

[0063] One skilled in the art can combine the above-mentioned embodiments with common sense to obtain more embodiments of the probe of the present application.

[0064] In some embodiments, the nucleic acid to be tested has 8 or fewer base mutations 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 embodiments, the annealing temperature of the probe when performing qPCR reaction with the nucleic acid to be tested is controlled to be 5°C-70°C.

[0067] As an example, in some specific embodiments, the annealing temperature of the probe when performing 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 a pathogenic microorganism.

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

[0070] In some embodiments, the nucleic acid to be tested according to the present application can be derived from various clinical sample types, such as blood samples (whole blood, plasma, serum), body fluid samples (cerebrospinal fluid, urine, pleural effusion), tissue samples (biopsy tissue, surgical resection tissue), secretion and excretion samples (sputum, feces, vomit), and other samples (hair, nails, interstitial fluid).

[0071] In some embodiments, the nucleic acid to be tested according to the present application can also be derived from food samples, environmental samples, crop samples, and the like.

[0072] In some embodiments, the sample to be tested or target nucleic acid is derived from a microorganism, a plant, or an animal (e.g., a mouse).

[0073] In some embodiments, the sample to be tested or target nucleic acid is derived from a human.

[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 surfactin sodium, 50-100 mmol / L of Tris-HCl at pH 8.0±0.5, and 0.5× SYBR Green I.

[0075] In some 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 surfactin sodium.

[0076] In some 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 of Tris-HCl at pH 8.0±0.5 (e.g., 7.5, 7.8, 8.0, 8.2, or 8.5).

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

[0078] In some embodiments, the qPCR reaction contains 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 probe in the reaction system.

[0079] In some embodiments, the qPCR reaction contains the following components in the reaction system per 20 μL: 1× RapidTaq Master Mix, 0.5× SYBR Green I, 50-100 mmol / L (preferably 100 mmol / L) Tris-HCl pH 8.0±0.5, 2-4 vol% (preferably 3 vol%) DMSO, 16-62.5 ng / μL (preferably 31 ng / μL) surfactin sodium, 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 balance being water.

[0080] In some embodiments, the qPCR reaction has the following conditions: 1) 95 ℃, 3 min; 1 cycle; 2) 95 ℃, 5 s; annealing (temperature as described above), 1 s; 69-70 ℃ (preferably 70 ℃), 1 s; 40 cycles.

[0081] The detection method of the present application can complete detection within 1 h, which is about 1 / 2-1 / 3 of the time of the traditional Taqman qPCR method, and meets the requirement of timeliness of detection results in large-scale detection scenarios.

[0082] All steps in the detection method of the present application can be implemented by human operation, by device, or by a combination of human operation and device.

[0083] The detection method of the present application can be based on diagnostic purposes or non-diagnostic purposes. In some embodiments (e.g., when based on diagnostic purposes), some steps (e.g., steps directly or indirectly related to diagnostic results) or all steps of the detection method can be implemented by a device.

[0084] In the specific implementation, the above-mentioned embodiments can be combined by those skilled in the art in combination with common sense to obtain more embodiments of the detection method of the present application.

[0085] The present application also provides a computer device comprising a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to realize the steps of the detection method based on FRET effect.

[0086] The present application also provides a computer readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to realize the steps of the detection method based on FRET effect.

[0087] The present application also provides a computer program product comprising computer program instructions, wherein the computer program instructions are executed by a processor to realize the steps of the detection method based on FRET effect.

[0088] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the art, or according to the conditions suggested by the manufacturer or according to the known experimental methods in the art.

[0089] In the following specific examples, the measurement parameters of the raw material components may, without specific instructions, have slight deviations within the weighing accuracy range. The temperature and time parameters allow for acceptable deviations caused by the instrument testing accuracy or operation accuracy.

[0090] Example 1 Distance relationship of Qian Hybridization probe intramolecular FRET effect

[0091] As shown in Table 1, six detection mousepox virus (MPV) Qian Hybridization probe molecules were designed respectively. Ectromelia virus) with stem region sequence of 7 fixed base pairs (5'-TCAGGCG-3' and 5'-CGCCTGA-3') and loop region sequence (probe specificity variation part) of 38 bps, 33 bps, 28 bps, 23 bps, 18 bps and 13 bps respectively. All the probe designs were outsourced to Suzhou Qianhybridization Biotechnology Co., Ltd. for synthesis. Under the condition of no addition of Taq enzyme and substrate, qPCR reactions were performed with different Qian Hybridization probes, with 3 replicates for each reaction. BioRad C1000 Touch Thermal Cycler / CFX96TM REAL-TIME SYSTEM was used for qPCR instrument, and the qPCR reaction system is shown in Table 2, and the qPCR reaction conditions are shown in Table 3. The relative fluorescence units of qPCR reaction Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated, and the calculation results are shown in Figure 2 .

[0092] Table 1 Qian Hybridization probe information of mousepox virus

[0093]

[0094] Table 2 qPCR reaction system without addition of Taq enzyme and substrate

[0095]

[0096] Table 3 qPCR reaction conditions

[0097]

[0098] The present application ingeniously uses the change of annealing temperature to control the opening and closing degree of Qian Hybridization probe under the condition of no addition of Taq enzyme and substrate, thereby changing the distance between the fluorescent reporter group and the fluorescent quencher group, and real-time monitoring the intensity of fluorescent signal released by Qian Hybridization probe under different opening and closing degrees, thereby confirming that the energy transfer efficiency of intramolecular FRET effect (E) is not a six-fold dependent relationship with the distance between the donor and the acceptor. E ) and the distance between the donor and the acceptor. Figure 2As shown, in the case of constant loop sequence length, the detected relative fluorescence units do not change significantly as the annealing temperature increases from 5°C to 35°C, at which time the complementary strand of the stem region of the Qian Hybridization probe is in a closed state, and the detected fluorescence signal is a background signal; when the annealing temperature increases to 45°C-75°C, the complementary strand of the stem region of the Qian Hybridization probe gradually opens under the action of high temperature, and the distance between the fluorescence reporter group and the fluorescence quenching group gradually increases, so that even if there is no substrate in the reaction system that is complementary to the probe, the relative fluorescence units will still show an upward trend as the annealing temperature increases; when the annealing temperature increases to above 85°C, the detected relative fluorescence units are basically stable, at which time the stem region of the Qian Hybridization probe is completely denatured, its conformation changes to a linear molecule, and the fluorescence signal is completely released. It can be seen that when the Qian Hybridization probe is in the "intermediate state" between complete closing and complete opening, it will still release a fluorescence signal, and the fluorescence intensity will linearly increase with the increase in the opening degree of the probe. This fact is contrary to the known FRET effect, which makes the FRET effect extremely sensitive to small changes in distance. As the probe gradually opens, the fluorescence signal will first be in a quenched state, and until the distance between the fluorescence reporter group and the fluorescence quenching group increases to a certain critical value, the energy transfer efficiency will suddenly drop, resulting in the sudden release of a large amount of fluorescence signal, rather than a stable linear growth relationship. R -6 The distance between the fluorescence reporter group and the fluorescence quenching group will make the FRET effect extremely sensitive to small changes in distance. As the probe gradually opens, the fluorescence signal will first be in a quenched state, and until the distance between the fluorescence reporter group and the fluorescence quenching group increases to a certain critical value, the energy transfer efficiency will suddenly drop, resulting in the sudden release of a large amount of fluorescence signal, rather than a stable linear growth relationship.

[0099] In addition, compared with the annealing temperature of 5℃, when the annealing temperature reached 95℃, the relative fluorescence units of V1KG-38 probe increased by 167.54%, the relative fluorescence units of V1KG-33 probe increased by 202.17%, the relative fluorescence units of V1KG-28 probe increased by 197.98%, the relative fluorescence units of V1KG-23 probe increased by 158.03%, the relative fluorescence units of V1KG-18 probe increased by 139.28%, and the relative fluorescence units of V1KG-13 probe increased by 119.06%. It can be seen that under the condition of the same increase of annealing temperature, the length of the loop region sequence of Qian Hybridization probe is different, and the intensity of the released fluorescence signal also has a large difference. In summary, within the annealing temperature range of 5℃-95℃, when the annealing temperature is constant, the longer the loop region sequence of Qian Hybridization probe, the greater the relative fluorescence units detected, and the change of relative fluorescence units is caused by the structure of Qian Hybridization probe itself. Since Qian Hybridization probe will make continuous random Brownian motion during the qPCR reaction, at this time the longer the loop region sequence, the larger the "ring", and the more unstable the spatial conformation of the probe during Brownian motion, resulting in fluctuations in FRET effect, thereby generating more fluorescence signals. In addition, Brownian motion is temperature-dependent, the higher the temperature, the more intense the Brownian motion, and this phenomenon is most obvious when the annealing temperature is increased to 45℃. Within the annealing temperature range of 45℃-95℃, the relative fluorescence units of Qian Hybridization probe present a typical stepwise increase with the increase of the length of the loop region sequence, that is, V1KG-38>V1KG-33>V1KG-28>V1KG-18>V1KG-13, and this stepwise change trend still exists when the molecular conformation of Qian Hybridization probe changes to linear molecule.

[0100] Example 2: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequences under the annealing temperature of 5℃

[0101] 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, respectively. The Qian Hybridization probe was designed as in Example 1. After the design of all probes and ssDNAs, they were synthesized by Suzhou Jinyuizhi Biotechnology Co., Ltd. Different Qian Hybridization probes were mixed with synthesized ssDNAs to perform qPCR reaction under the condition of no addition of Taq enzyme, and ddH2O was added instead of ssDNA as negative control, 3 replicates for each reaction. The qPCR instrument was BioRad C1000 Touch Thermal Cycler / CFX96TM REAL-TIME SYSTEM with 5 fluorescence channels, the qPCR reaction system was as shown in Table 5, and the qPCR reaction conditions were as shown in Table 6. The relative fluorescence units of the qPCR reaction Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated, and the calculation results were as shown in Table 7. Figure 3 .

[0102] Table 4 ssDNA sequence

[0103]

[0104] Table 5 qPCR reaction system without addition of Taq enzyme

[0105]

[0106] Table 6 qPCR reaction conditions

[0107]

[0108] Example 3: Luminescence characteristics of Qian Hybridization probe in detection of mutant sequence at 15℃ annealing temperature

[0109] Different Qian Hybridization probes were mixed with synthesized ssDNAs to perform qPCR reaction under the condition of no addition of Taq enzyme, and ddH2O was added instead of ssDNA as negative control, 3 replicates for each reaction, the Qian Hybridization probe was designed as in Example 1, and the ssDNA was designed as in Example 2. Except that the annealing temperature was changed to 15℃, the qPCR instrument, qPCR reaction system and qPCR reaction conditions were the same as in Example 2. The relative fluorescence units of the qPCR reaction Rox fluorescence channel at the end of each amplification cycle were recorded, and the average relative fluorescence units were calculated, and the calculation results were as shown in Table 8. Figure 4 .

[0110] Example 4: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 25℃ annealing temperature

[0111] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 25℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 5 .

[0112] Example 5: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 35℃ annealing temperature

[0113] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 35℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 6 .

[0114] Example 6: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 45℃ annealing temperature

[0115] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 45℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 7 .

[0116] Example 7: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 55℃ annealing temperature

[0117] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 55℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 8 .

[0118] Example 8: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 60℃ annealing temperature

[0119] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 60℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 9 .

[0120] Example 9: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 65℃ annealing temperature

[0121] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 65℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 10 .

[0122] Example 10: Luminescence characteristics of Qian Hybridization probes in detecting mutant sequences at 70℃ annealing temperature

[0123] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 70℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 11 .

[0124] Example 11: Luminescence characteristics of Qian Hybridization probes in detecting mutant sequences at 75℃ annealing temperature

[0125] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 75℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 12 .

[0126] Example 12: Luminescence characteristics of Qian Hybridization probes in detecting mutant sequences at 85℃ annealing temperature

[0127] Under the condition of no addition of Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA to perform qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 85℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 13 .

[0128] Example 13: Luminescence characteristics of Qian Hybridization probe in detecting mutant sequence at 95℃ annealing temperature

[0129] Under the condition without adding Taq enzyme, different Qian Hybridization probes were mixed with synthetic ssDNA respectively to carry out qPCR reaction, and ddH2O was added to replace ssDNA as negative control, each reaction had 3 duplicate holes, the design of Qian Hybridization probe was the same as that in Example 1, and the design of ssDNA was the same as that in Example 2. Except that the annealing temperature was changed to 95℃, the qPCR instrument, qPCR reaction system and qPCR reaction condition were the same as those in Example 2. The relative fluorescence units of qPCR reaction Rox fluorescence channel after each amplification cycle was recorded, and the average relative fluorescence units were calculated, and the calculation results were as follows Figure 14 .

[0130] As Figures 3 to 14As shown, at annealing temperature of 5-35℃, Qian Hybridization probe is in the off state without ssDNA. When the length of Qian Hybridization probe loop region sequence is 13 bps, even if the ssDNA completely complementary to the loop region sequence is added in the reaction system, the relative fluorescence units detected are only about 3% higher than the negative control. This may be because the loop region sequence is too short, and the intermolecular force formed by pairing and combining with the substrate is smaller than the intramolecular binding force of the stem region. On the other hand, the hydrogen bond 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 fluorescence reporter group and the fluorescence quencher group, so only weak fluorescence signal can be generated. Since the vertical distance between adjacent base pairs in a DNA molecule is 0.34 nm, the straight-line distance between the fluorescence reporter group and the fluorescence quencher group should be less than the vertical distance after the loop region sequence pairing and combining is "straightened" (0.34 nm x 13 bps = 4.42 nm). Therefore, it can be inferred that when Qian Hybridization probe is in the complete off state, the straight-line distance between the fluorescence reporter group and the fluorescence quencher group is actually less than 5 nm. When the loop region sequence is increased to 18-38 bps, specific hybridization with ssDNA without mutation will cause the stem region to open to varying degrees, and the longer the loop region sequence, the higher the degree of stem region opening, and the stronger the released fluorescence signal. Therefore, the straight-line distance between the fluorescence reporter group and the fluorescence quencher group is 6-13 nm (0.34 nm x 18 bps; 0.34 nm x 38 bps), which can produce a fluorescence signal significantly higher than the background. In addition, as the number of base mutations in ssDNA linearly increases to 1, 3, 5, and 7, the relative fluorescence units detected also show a similar linear downward trend, and for ssDNA with 7 base mutations, the fluorescence intensity detected is still higher than the negative control. At this time, Qian Hybridization probe has high mutation tolerance.However, as the annealing temperature gradually increases, at the annealing temperature of 45-75°C, the Qian Hybridization probe is in the gradually opening "intermediate state", the length of the loop region sequence gradually enhances the influence on the mutation resistance of the Qian Hybridization probe, when the annealing temperature is 60°C, only the Qian Hybridization probes with 38 bps, 33 bps and 28 bps loop region sequences can bind with the ssDNA with 7 base mutations to generate a fluorescence signal higher than the negative control, when the annealing temperature is 65°C, only the Qian Hybridization probes with 38 bps and 33 bps loop region sequences can bind with the ssDNA with 7 base mutations to generate a fluorescence signal higher than the negative control, when the annealing temperature is 70°C, only the Qian Hybridization probe with 38 bps loop region sequence can bind with the ssDNA with 5 base mutations to generate a fluorescence signal higher than the negative control, and when the annealing temperature is 75°C, all the Qian Hybridization probes cannot bind with the mutated ssDNA to generate a fluorescence signal higher than the negative control. It can be seen that, as the annealing temperature increases, the rigidity stress formed after the Qian Hybridization probe loop region sequence binds with the ssDNA will act on the stem region together with the high temperature, at this time, the higher the temperature is, the higher the opening degree of the stem region is, if the loop region sequence is shorter and the number of mutated bases of the ssDNA is more at this time, the rigidity stress effect formed in the mismatch hybridization is less than the opening degree of the stem region caused by the high temperature, so that the fluorescence intensity higher than the background signal cannot be generated, resulting in detection failure. When the annealing temperature increases to above 85°C, the stem region of the Qian Hybridization probe has been completely opened, at this time, even if the length of the loop region is 38 bps and completely matches with the ssDNA without mutation, the fluorescence intensity higher than the background signal also cannot be generated, which indicates that the straight line distance between the fluorescence reporter group and the fluorescence quenching group has exceeded the influence range of the FRET effect at this time, from which it can be inferred that the straight line distance between the fluorescence reporter group and the fluorescence quenching group of the Qian Hybridization probe in the completely open state should be greater than 13 nm (0.34 nm*38 bps). Therefore, according to the present application. Figures 3 to 14 The linearly decreasing trend of the fluorescence intensity with the increase of the number of mutated bases of the substrate at the specific annealing temperature is shown, and it is deduced that when the distance between the donor and the acceptor is greater than 6 nm and less than 13 nm, the energy transfer efficiency of the intramolecular FRET effect is R ) formula: E

[0131]

[0132] wherein, k ​The slope R 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 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.

[0133] Example 14: 2D qPCR reaction system for mousepox virus ( Ectromelia virus Detection capability of mutant strains

[0134] 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.

[0135] Table 7. Detection of mousepox virus in 2D qPCR reaction system ( Ectromelia virus QianHybridization probe

[0136]

[0137] Table 8 Mousepox virus ( Ectromelia virus 2D qPCR reaction system for sequence fragments

[0138]

[0139] Table 9 Mousepox virus ( Ectromelia virus Detection primer pairs for sequence fragments

[0140]

[0141] Table 10 Mousepox virus ( Ectromelia virus 2D qPCR reaction conditions for sequence fragments

[0142]

[0143] 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.

[0144] Comparative Example 1: Traditional probe-based qPCR reaction system against mousepox virus ( Ectromelia virus Detection capability of mutant strains

[0145] 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 .

[0146] like Figure 17As shown, compared with the present application, using the conventional Taqman hydrolysis probe cannot identify any mutant strains, and only a strong enough fluorescence signal is detected in the positive control (V1-0), and when 1-8 base mutations occur in the sequence fragments of the oligonucleotide, no fluorescence signal is detected. This is because the Taqman hydrolysis probe can only specifically bind to the unmutated target sequence, and after binding, it will be hydrolyzed by the 5'→3' exonuclease activity of Taq enzyme, so that the fluorescence reporter group and the fluorescence quenching group are separated to a distance of more than 10 nm, the FRET effect is terminated, and the fluorescence signal is released; but in the face of mutant strains, the Taqman hydrolysis probe will be difficult to bind to the mutated target sequence, or after binding, it cannot be hydrolyzed by the 5'→3' exonuclease activity of Taq enzyme, so that the probe remains intact, at this time the distance between the fluorescence reporter group and the fluorescence quenching group is less than 10 nm, so no fluorescence signal is generated, which leads to detection failure.

[0147] The above results fully show that the 2D qPCR system of the present application breaks through the theory of FRET effect, reveals the energy transfer law of intramolecular FRET effect, and solves the dilemma of variation escape and detection failure of the traditional qPCR method. Not only has strong anti-variation ability, but also can make early warning of early evolution of viruses in the 2D matrix coordinate system through the change of fluorescence intensity, so as to cope with the off-target risk in the detection of high-variation pathogens. It has great significance for real-time monitoring of pathogen mutation during the epidemic period and formulating prevention and control strategies.

[0148] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A method for detecting based on FRET effect, comprising: obtaining energy transfer efficiency E of intramolecular FRET effect; determining linear distance R between 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 energy transfer efficiency E and linear distance R, respectively, and the first functional relationship is determined according to the linear relationship between the energy transfer efficiency E and the reciprocal of the linear distance R of the intramolecular donor and acceptor -1 . 2.The method for detecting based on FRET effect according to claim 1, comprising: obtaining relative fluorescence unit F of the molecule; determining linear distance R between intramolecular donor and acceptor according to 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 relative fluorescence units F and linear distance R, respectively, and the second functional relationship is determined according to a linear relationship between energy transfer efficiency E and the inverse of relative fluorescence units F -1 and the inverse of linear distance R -1 respectively. 3.The method for detecting based on FRET effect according to claim 1, comprising: obtaining relative fluorescence unit F of the molecule; determining internal conformation of the molecule according to the relative fluorescence unit F and a first threshold range; wherein the internal conformation of the molecule is different, the intramolecular donor and acceptor have different linear distances R; the linear distance R satisfies 6 nm < R < 13 nm; The first threshold range is determined based on a method of determining a threshold range of relative fluorescence units F associated with the internal conformation of the molecule based on a linear relationship of the energy transfer efficiency E and the inverse of the relative fluorescence units F -1 and the inverse of the straight-line distance R -1 and the association of the straight-line distance R with the internal conformation of the molecule. 4.The method for detecting based on FRET effect according to claim 1, comprising: obtaining relative fluorescence unit F of the molecule when the molecule is combined with a to-be-detected substance; determining matching degree of the to-be-detected substance according to the relative fluorescence unit F and a second threshold range; wherein the matching degree of the molecule with the to-be-detected substance 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 a method of determining a threshold range of relative fluorescence units F associated with the matching degree of the substance to be measured according to a linear relationship of energy transfer efficiency E and inverse of relative fluorescence units F -1 and inverse of straight line distance R -1 , and the relevance of straight line distance R to the matching degree of the substance to be measured.

5. The detection method based on FRET effect according to claim 4, wherein, the molecule is a probe, and the to-be-detected substance is a to-be-detected nucleic acid; the probe comprises, from 5' end to 3' end, a fluorescent reporter group, a stem region sequence, an alkyl spacer, a loop region sequence, an alkyl spacer, a stem region sequence and a fluorescent quencher group; wherein the two stem region sequences can be complementary to each other, so that the probe forms a hairpin structure; the loop region sequence comprises a region complementary to the target nucleic acid; the alkyl spacer is a hydrophobic straight-chain alkyl spacer.

6. The detection method based on FRET effect according to claim 4, wherein, the molecule is a probe, and the to-be-detected substance is a to-be-detected nucleic acid; the obtaining of the relative fluorescence unit F of the molecule when the molecule is combined with the to-be-detected substance specifically comprises: obtaining the relative fluorescence unit of the fluorescent channel after each amplification cycle of the qPCR reaction of the probe and the to-be-detected nucleic acid, and calculating the average relative fluorescence unit as the relative fluorescence unit F.

7. The detection method based on FRET effect according to claim 4, wherein, the molecule is a probe, and the to-be-detected substance is a to-be-detected nucleic acid; the determining of the matching degree of the to-be-detected substance according to the relative fluorescence unit F and the second threshold range specifically comprises: when the relative fluorescence unit F is higher than the background signal and lower than the average relative fluorescence unit measured when the molecule is combined with the target nucleic acid, it is judged that the to-be-detected nucleic acid has at least one base mutation compared with the target nucleic acid.

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

9. The detection method based on FRET effect according to claim 5, wherein, the stem region sequence comprises any one group of sequences in 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 FRET effect according to claim 5, wherein, The length of the loop sequence is 23-43 bps.

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

12. The detection method based on FRET effect according to claim 5, wherein, The hydrophobic linear alkyl spacer is C3 Spacer or C6 Spacer.

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

14. The detection method based on FRET effect according to claim 5, wherein, The annealing temperature of the probe in the qPCR reaction with the nucleic acid to be detected is 5-70℃.

15. A computer apparatus comprising a memory, a processor, and a computer program stored on 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 1-14.

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

17. A computer program product comprising computer program instructions, wherein, The computer program instructions are executed by the processor to implement the steps of the detection method based on the FRET effect according to any one of claims 1-14.

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