H1N1 virus hypersensitive nucleic acid detection system of CRISPR-Cas12a system as well as kit and application of H1N1 virus hypersensitive nucleic acid detection system

Abstract

The invention discloses a CRISPR-Cas12a system H1N1 virus hypersensitive nucleic acid detection system and a kit and application thereof, the CRISPR-Cas12a system H1N1 virus hypersensitive nucleic acid detection system comprises a Cas12a recognition system, an allosteric regulation component, a signal amplification component and a report system, the system reduces background noise through allosteric inhibition, improves sensitivity through dual signal amplification, and realizes hypersensitive detection of an H1N1 virus M1 gene in combination with ErCas12a protease. The method has the advantages of low detection lower limit, no dependence on natural PAM sequence, less than 1 hour of time consumption and the like, and the sensitivity and the specificity are superior to those of the existing RT-qPCR detection method. By adjusting crRNA and probe sequences, the platform disclosed by the invention can be expanded for portable rapid detection of various respiratory pathogens, and is particularly suitable for primary medical treatment and field emergency screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomolecular medical detection, in particular to a H1N1 virus hypersensitive nucleic acid detection system based on CRISPR-Cas12a system, and also relates to a kit constructed by the system and application. BACKGROUND

[0002] Influenza A H1N1 virus is one of the main pathogens causing seasonal influenza and pandemic, with the characteristics of strong infectivity and rapid transmission. Timely and accurate detection of H1N1 virus is of great significance for epidemiological monitoring and clinical diagnosis and treatment. However, the existing H1N1 detection methods have many limitations. For example, the virus culture method takes a long time and has high requirements for laboratory conditions; the antigen rapid detection is simple to operate but has low sensitivity and high false negative rate; the nucleic acid detection (such as reverse transcription quantitative PCR, RT-qPCR) has high sensitivity and specificity, but requires expensive equipment and professional operation, and the detection period is relatively long, which is not conducive to on-site instant diagnosis. In resource-limited or primary medical environment, there is an urgent need for a rapid, portable and hypersensitive H1N1 virus nucleic acid detection method to realize instant diagnosis and large-scale screening of early infection.

[0003] The CRISPR / Cas12a system, which has emerged in recent years, provides a new tool for molecular diagnosis. After recognizing a specific target nucleic acid, the Cas12a protein not only cuts the target sequence itself, but also activates its non-specific single-stranded DNA endonuclease activity (collateral cleavage), which can cleave the surrounding reporter probe with a fluorescent label, thereby amplifying a single target recognition event into a detectable signal. By combining Cas12a detection with isothermal nucleic acid amplification technology, highly sensitive detection of pathogen nucleic acids can be achieved without the need for complex instruments. For example, recombinase aided amplification (RAA) can efficiently amplify specific DNA sequences in about 37°C for 30 minutes, has low equipment requirements, and is suitable for rapid on-site detection. Previous studies have combined RAA with CRISPR-Cas12a for pathogenic microorganism nucleic acid detection. However, the traditional CRISPR-Cas12a highly sensitive detection system still faces some problems: first, non-specific background signals may occur when high sensitivity is pursued, or false positives may occur due to amplification product aerosol contamination; second, Cas12a recognition of the target requires a PAM sequence (such as TTTV), which limits direct detection of targets that do not contain compatible PAM sites. To improve detection specificity, sensitivity, and target universality, researchers in the field have proposed using an allosteric regulation mechanism to control Cas12a enzyme activity in a target-dependent manner, and designing an engineered probe system to amplify the signal. For example, by designing special structure crRNA or adding additional aptamer nucleic acid probes, Cas12a can be kept inactive without the correct target, and only when the target is bound can Cas12a trigger collateral cleavage activity, thereby significantly reducing background noise and improving detection reliability. At the same time, the introduction of split crRNA (bipartite-crRNA) nucleic acid system, after the first activation of Cas12a, the conformation changes, and the free split nucleic acid component can competitively replace the original crRNA, and activate Cas12a again to trigger the second round of reporter probe cleavage, realizing the cascade amplification of signal. Therefore, the combination of double-stranded DNA pre-activated Cas12a allosteric regulation and bipartite-crRNA competitive replacement amplification mechanism is expected to construct a rapid, highly sensitive, and specific H1N1 virus nucleic acid detection platform for on-site rapid diagnosis. SUMMARY

[0004] Therefore, one of the purposes of the present application is to provide an H1N1 virus highly sensitive nucleic acid detection system of CRISPR-Cas12a system; the second purpose of the present application is to provide an H1N1 virus nucleic acid detection method for non-diagnostic purposes; the third purpose of the present application is to provide a kit for detecting H1N1 virus.

[0005] To achieve the above purposes, the present application provides the following technical solutions: 1. A H1N1 virus hyper-sensitive nucleic acid detection system of CRISPR-Cas12a system, the system comprising: (a) a Cas12a recognition system comprising a Cas12a protease and a specific crRNA; the Cas12a protease has PAM recognition ability; the crRNA comprises a guide sequence complementary to a H1N1 virus target nucleic acid and a stem loop structure; (b) an allosteric regulation component comprising a pair of complementary oligonucleotide sequences; wherein one oligonucleotide hybridizes with a partial sequence of crRNA to form a double-stranded structure in the absence of target nucleic acid, thereby inhibiting the cleavage activity of Cas12a protease, and the oligonucleotides are both 5' phosphorylated for Cas12a to recognize the PAM sequence at the end of the complementary oligonucleotide sequence after binding; (c) a free nucleic acid probe system comprising a bipartite-crRNA combination and a reporter probe; the bipartite-crRNA combination consists of a bipartite-crRNA handle fragment, a bipartite-crRNA spacer fragment and an activator oligonucleotide, the bipartite-crRNA handle is an RNA or DNA oligonucleotide, which contains a partial straight hairpin structure of crRNA; the bipartite-crRNA spacer is an oligonucleotide, the sequence of which is partially complementary to the H1N1 target sequence; the activator is a short oligonucleotide complementary to the bipartite-crRNA spacer; after the Cas12a protease is activated by the target nucleic acid for the first time, the conformation changes, competes with the bipartite-crRNA combination for binding, replaces the original crRNA binding to Cas12a, and activates the enzyme activity of Cas12a again, thereby triggering the secondary cleavage of the free reporter probe to amplify the signal; the reporter probe is labeled with a detectable label, and when the reporter probe is cleaved by the activated Cas12a protease, a detectable signal is generated.

[0006] Preferably, the crRNA specifically recognizes a conserved fragment of the M1 gene of H1N1 virus, and the sequence is shown as SEQ ID NO. 1.

[0007] Preferably, one sequence of the allosteric regulation component is complementary to the sequence of crRNA, and the length is 9-15 bp, and the nucleotide sequence is shown as SEQ ID NO. 2 and SEQ ID NO. 3. Preferably, the signal amplification component consists of the nucleotide sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

[0008] Preferably, the free reporter probe is an oligonucleotide probe with a length of 5-10 nt, each end of which is labeled with two FAM fluorescent groups, and a BHQ1 quenching group and a Biotin molecule are labeled at the middle through a thymine site, and the sequence is as follows: Biotin-TTATGT(FAM)TGTATGT(FAM)TGTATT(BHQ1)TTAT(FAM)TTATG-FAM.

[0009] Preferably, the Cas12a protease is an ErCas12a protease from Eubacterium rectale.

[0010] 2. A method for detecting H1N1 virus nucleic acid for non-diagnostic purposes, using the detection system, the method comprising the following steps: (1) mixing the sample to be tested with the detection system; (2) incubating under conditions suitable for Cas12a protease reaction; (3) detecting the signal generated by the reporter system, wherein the presence of the signal indicates the presence of H1N1 virus target nucleic acid in the sample.

[0011] Preferably, the sample to be tested is a throat swab or nasal swab sample collected from a patient to be tested.

[0012] 3. A kit for detecting H1N1 virus, the kit comprising the detection system. Preferably, the kit further comprises a field nucleic acid extraction / cleavage reagent for rapidly releasing viral nucleic acid from throat swab, sputum and other samples; a portable fluorescent signal detection device or a handheld blue light excitation light source for real-time monitoring or naked eye observation of fluorescent signal; and a nucleic acid detection test strip reading device for interpreting results in a lateral flow chromatography mode.

[0013] The application has the beneficial effects that the application discloses an H1N1 virus hypersensitive nucleic acid detection system of a CRISPR-Cas12a system, the system uses a double-stranded DNA molecule to pre-activate and allosterically regulate Cas12a, greatly reduces the background signal leakage, and realizes the direct detection of the RNA target without amplification; meanwhile, a cascade displacement amplification mechanism driven by crRNA affinity reversal is introduced, which greatly improves the detection sensitivity. Compared with the existing CRISPR-Cas12a detection scheme, the platform has three cross-generation advantages: first, the detection lower limit of the viral RNA reaches the attomole order of magnitude concentration, and the sensitivity is improved by more than one order of magnitude compared with the previous Cas12a method requiring pre-amplification; second, the target nucleic acid can be detected without relying on the natural PAM sequence, which expands the target application range of Cas12a; third, the whole process of "sample in-result out" can be completed within 60 minutes without complex equipment such as a thermal cycler, and can be operated only by relying on a constant temperature device, and the single detection cost is low, which is convenient for grassroots and on-site application. The minimum detection limit of the H1N1 virus RNA of the platform can reach the attomole level (about 10^-18 M order of magnitude), and the sensitivity is better than that of the conventional RT-qPCR method; the detection can be completed within 60 minutes and no false positive caused by non-target virus occurs, and the specificity can reach about 93% or more. In addition, since the sequences of the detection elements (such as crRNA and report probes) can be flexibly designed and replaced according to the target, the platform has good universality and scalability, and in addition to H1N1 virus, it can also be used for detecting nucleic acids of other respiratory pathogens by replacing specific sequences. In summary, the application provides a new nucleic acid detection strategy which is rapid, simple, hypersensitive and specific, and is particularly suitable for early diagnosis and large-scale screening of H1N1 and other respiratory pathogens in grassroots medical care and on-site emergency. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to make the purpose, technical scheme and beneficial effects of the application more clear, the application provides the following drawings for description: Figure 1 The figure is a schematic diagram of the overall process of the H1N1 virus CRISPR-Cas12a hypersensitive detection platform (a: the process of extracting H1N1 virus RNA from a clinical sample and adding the detection system of the application for detection is shown; b: the principle of the detection mechanism is shown, which shows that the ErCas12a / complete crRNA is pre-activated by a double-stranded DNA activator (dsDNAa) to form an inactive complex, which is activated and triggers a bipartite-crRNA competition displacement cascade amplification cycle when there is an RNA target, and finally produces an active product to cut the report probe to output a signal); Figure 2Characterization and optimization results of the properties of double-stranded DNA activator (a: design of ssDNA activator and dsDNA activator complementary to the proximal region of crRNA seed region (length range 9-15 nt); b: comparison of background signal leakage intensity when pre-activating ErCas12a / crRNA complex with activators of different lengths: when the length of the activator complementary to the crRNA is ≤14 nt, ErCas12a has no trans-cleavage activity at all; and at the same length, the background fluorescence leakage using dsDNA activator is significantly lower than that of ssDNA activator (more than an order of magnitude lower), which shows that the activator with double-stranded structure can effectively inhibit the leakage of non-specific enzyme activity due to its stronger binding rigidity); Figure 3 Experimental results of the three-mode signal output capability of the 4FAM compatible reporter probe in the application (a: comparison of fluorescence signal intensity of DF-Cas12a system with and without bipartite-crRNA combination, it is found that the signal is significantly enhanced (X times, p<0.001) after introducing bipartite-crRNA, proving the contribution of cascade amplification to fluorescence signal; b-c: comparison of fluorescence intensity of different fluorescence label density probes, under the same target concentration, the end-point fluorescence intensity generated by 4FAM probe is significantly higher than that of 2FAM and 1FAM probes (p<0.001), and the overall signal intensity is positively correlated with the number of FAM labels; d: shows the visible fluorescence results of different probes under 470 nm blue light irradiation: the fluorescence generated by 4FAM probe is the brightest, followed by 2FAM, and 1FAM is almost invisible, the visible observation results are consistent with the quantitative fluorescence intensity; e: shows the lateral flow chromatographic test strip (LFA) detection results: 4FAM probe presents a clear and visible red band on the test line (T line) of the test strip, 2FAM probe only appears a very weak band, and 1FAM probe does not show signal on the T line (only the quality control line C line appears), it is speculated that 1FAM probe cannot migrate normally with gold nanoparticles due to the interference of BHQ1 quencher; the results show that at least two FAM groups are needed to generate recognizable signal in the LFA visualization mode; f: 4FAM compatible probe is directly compared with traditional single FAM probe: when using 4FAM probe, the fluorescence reading and test strip T line color development intensity are much better than those of traditional probe (single FAM-BHQ1 has no any signal for LFA, and single FAM-Biotin has weak signal for fluorescence detection), which fully embodies the innovation and advantages of 4FAM compatible probe in multi-mode reading; in summary, by innovatively designing 4FAM labeled compatible reporter probe, the application realizes three signal output modes of one-tube real-time fluorescence detection, naked-eye fluorescence direct reading and lateral flow test strip colorimetric, which greatly facilitates result interpretation and application expansion; Figure 4The graph shows the evaluation results of the detection platform of this invention for RNA and DNA target detection (ad: the sensitivity and specificity evaluation of the DF-Cas12a system for H1N1 influenza A virus RNA: a shows the fluorescence intensity after 30 minutes of reaction under gradient dilution of H1N1 virus RNA (100aM to 10 pM), showing a good dose-dependent relationship (R). 2 =0.97), which can stably quantify RNA copy number within this concentration range; b is the standard curve linear regression result, which shows an approximately 37.8-fold increase in sensitivity at low concentrations compared to the traditional CRISPR-Cas12a detection method (p<0.001); c is the specificity validation result, when the DF-Cas12a system was applied to RNA samples of common non-target respiratory viruses (such as IBV, RSV, rhinovirus, etc.), the fluorescence signals of all non-target samples were close to the negative control background level (ΔRFU<5%), and no cross-reactivity was observed; d examines the repeatability and accuracy of the system, with the same sample being tested 35 times repeatedly, and the fluorescence results were highly consistent with the RT-qPCR quantification results (R 2 =0.92), intra-batch coefficient of variation (CV) < 4.5%.

[0015] Figure 5 The figure shows a comparison of the results of on-site clinical sample testing using the DF-Cas12a system. (a: Consistency analysis of the results between the DF-Cas12a method of this invention and the hospital RT-qPCR method: In a blind test of 50 clinical pharyngeal swab samples, RT-qPCR confirmed 35 positive cases and 15 negative cases; when DF-Cas12a detection used RFU%>2.0 as the criterion for positivity, the positive concordance rate (PPA) with the RT-qPCR results was 91.4% (32 / 35), and the negative concordance rate (NPA) was 93.3%). (14 / 15), with an overall concordance rate of 92.0% and a Kappa value of 0.85; b: Detailed comparison of quantitative results of RT-qPCR and DF-Cas12a on various samples, including 3 weakly positive samples with RT-qPCR Ct values ​​>35 that were not detected by DF-Cas12a (false negatives), and 1 sample that was negative by RT-qPCR but co-infected with RSV was misjudged as positive by DF-Cas12a (false positive). The detection time of the method is no more than 60 minutes, and the sensitivity (91.4%) and specificity (93.3%) both meet the WHO's performance requirements for point-of-care testing (POCT), providing a broad and efficient solution for respiratory pathogens. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0017] Preparation of reagents for detection platform The main reagents required for the H1N1 virus detection platform provided in this example include: Cas12a protein, crRNA and related nucleic acid probes. The specific composition is as follows: Cas12a protease: Cas12a variant (ErCas12a) derived from human intestinal bacteria Eubacterium rectale is selected, which has high enzyme activity and loose PAM recognition ability. Recombinant purified protein can be purchased from commercial channels (for example, NEB company item number M0653T), and stored at -20°C before use.

[0018] crRNA preparation: The guide sequence (Spacer) of crRNA is designed according to the conserved region sequence of H1N1 virus genome, with a length of 20 nt, and a repeat straight hairpin structure (36 nt constant sequence) is added at its 3' end. The nucleotide sequence of the crRNA used in this example is as follows: 5'-UAAUUUCUACUAAGUGUAGAUGAUCAGCAUUAACGACCUCGGUUA-3' (SEQ ID NO. 1) (5' end is Spacer sequence, completely complementary to H1N1 M1 gene specific fragment). The crRNA can be synthesized by in vitro transcription or chemically synthesized by a biological company and purified by HPLC. Before use, the crRNA is dissolved in nuclease-free water to 10 μM, and aliquoted and stored at -80°C.

[0019] Double-stranded DNA activator (dsDNA activator): composed of a pair of complementary oligonucleotide sequences, wherein the sense strand: 5'-CGCTTTAGATCAGCATTA-3' (SEQ ID NO. 2) is completely complementary to the partial sequence (e.g. the first 12 nucleotides of the seed region) near the end of the crRNA Spacer, and the antisense strand: 5'-TAATGCTGATCTAAAGCG-3' (SEQ ID NO. 3) is complementary to the sense strand to form a double strand. Both oligonucleotides are treated with 5' end phosphorylation to allow Cas12a to recognize the pseudo-PAM sequence (e.g. 5'-TTTA) at the end of the short double-stranded DNA after binding. The length of the dsDNA activator in this example is 12 bp, which simulates binding to the seed region of crRNA, and can effectively form a pre-activated complex with ErCas12a without triggering background cleavage reaction. After synthesis, the complementary strands are mixed in equimolar, denatured at 95°C and slowly cooled to anneal into double-stranded, with a final concentration of 2 μM for standby.

[0020] Bipartite-crRNA assembly: including bipartite-crRNA handle fragment, bipartite-crRNA spacer fragment and activator oligonucleotide. The bipartite-crRNA handle is an RNA or DNA oligonucleotide with a length of ~20 nt, and its sequence contains part of the straight hairpin structure of Cas12a crRNA: 5'-UAAUUUCUACUAAGUGUAGAU-3' (SEQ ID NO. 4); the bipartite-crRNA spacer is an oligonucleotide with a length of ~20 nt: 5'-GUAGUGUAUGUGUAGUAUGUA-3' (SEQ ID NO. 5), and its sequence is partially complementary to the H1N1 target sequence (in this example, it is designed to be the same sequence as the crRNA spacer to target the H1N1 M1 gene fragment); the activator is a short oligonucleotide complementary to the bipartite-crRNA spacer: 5'-TACACATACACTAC-3' (SEQ ID NO: 6). In this example, the handle and spacer fragments are chemically synthesized RNA oligonucleotides, and the activator is a DNA oligonucleotide. Before use, they are dissolved in TE buffer to form 20 μM stock solution and stored at low temperature. When needed, they are mixed and added to the system at the required concentration. The "window" of the conformation formed after the first activation of Cas12a by the target is used to induce the reconfiguration of the bipartite-crRNA functional RNP and mediate the secondary amplification of the signal.

[0021] Reporter probe: a bifunctional reporter probe with multiple fluorescent groups is used to realize fluorescence and lateral flow chromatography double output. In this example, a 5nt long oligonucleotide probe is used, which is labeled with two FAM fluorescent groups at both ends (to improve the fluorescence intensity), and a BHQ1 quenching group and a Biotin molecule (for test paper capture) are labeled at the middle through a thymine site. Its sequence is recorded as SEQ ID NO: 7, and the specific sequence is as follows: Biotin-TTATGT(FAM)TGTATGT(FAM)TGTATT(BHQ1)TTAT(FAM)TTATG-FAM. The fluorescence of the complete probe is completely quenched by the close BHQ1, and no background signal is produced; once Cas12a is activated, its non-specific cutting action randomly cuts any bond of the probe, so that part of the FAM is released from the quenching effect of the probe and releases fluorescence. The above probe is synthesized and purified by a biological company according to the sequence, and is prepared into 10 μM before use.

[0022] In addition to the core reagents described above, a Cas12a enzyme reaction buffer (e.g. NEB 2.1 buffer or a self-optimized 10x Cas12a Reaction Buffer), nuclease-free water, and a portable fluorescence reader or a handheld blue light excitation light source and lateral flow chromatographic test strip (Tiosbio® nucleic acid detection test strip is selected in this embodiment, which is pre-coated with anti-FAM antibody on the detection line and streptavidin on the quality control line, which can be combined with Biotin) for result interpretation are also required. All reagents are equilibrated to room temperature and gently mixed before use.

[0023] Example 2, Fluorescent Real-Time Detection of H1N1 Virus RNA (1) Reaction system configuration: Add the following components in a nuclease-free PCR tube to constitute a 20 μL reaction system: ErCas12a protein 1 μL (1 μM stock solution, final concentration 100 nM), 10x Cas12a reaction buffer 2 μL (final concentration 1x), crRNA liquid 1 μL (10 μM stock solution diluted to 600 nM, final concentration about 60 nM), dsDNA activated probe 1 μL (2 μM, final concentration 20 nM), bipartite-crRNA handle 0.5 μL (20 μM diluted to 200 nM, final concentration about 10 nM), bipartite-crRNA spacer 0.5 μL (20 μM diluted to 200 nM, final concentration about 10 nM), activator probe 0.5 μL (20 μM diluted to 200 nM, final concentration about 10 nM), 4FAM compatible reporter probe 2 μL (10 μM diluted to 4 μM, final concentration 400 nM), and the RNA sample to be tested 2 μL (containing H1N1 virus RNA or negative control RNA in the sample). Finally, make up the system to 20 μL with nuclease-free water, gently mix and collect the droplets by instantaneous centrifugation.

[0024] (2) Reaction condition: The reaction tube was placed in a 37°C constant temperature fluorescence detector for incubation for 30 minutes, and the fluorescence signal of the FAM channel was read once every 30 seconds. The real-time fluorescence intensity collected by the instrument increased with the reaction time, and if the target H1N1 RNA existed, the fluorescence signal was significantly higher than that of the negative control after about 10 minutes of reaction, and the background was effectively suppressed at a very low level. After 30 minutes of reaction, the percentage index of fluorescence intensity (RFU%) = (sample fluorescence / negative control fluorescence) x 100% was calculated for each reaction curve. According to the pre-established judgment threshold (in this embodiment, RFU% = 200%, i.e. 2.0 is the positive threshold), the presence or absence of the target was determined: RFU% ≥ 2.0 is positive, and less than 2.0 is negative. The results can also be verified by observing the fluorescence change with the naked eye: after 10 minutes of reaction, the PCR tube was placed under a portable blue light exciter (470 nm) for irradiation, and the positive sample emitted visible fluorescence with the naked eye, while the negative control had no obvious fluorescence.

[0025] (3) Result example: The fluorescence detection method was tested with H1N1 virus RNA gradient samples with known copy numbers, and it was found that RNA with a minimum concentration of 100 aM (~10-16 M) could stably produce a fluorescence signal higher than the background, realizing quantitative detection, and the dynamic range spanned nearly 5 orders of magnitude. According to the 3-sigma criterion, the theoretical LOD of the system for H1N1 RNA is about tens of aM, equivalent to <10^3 copies of RNA per reaction tube, which is about 1 order of magnitude more sensitive than the traditional Cas12a method relying on RT-RPA pre-amplification. Non-target RNA (such as other respiratory virus RNA) did not cause significant fluorescence enhancement, proving that the system has good specificity. Repeated experiments showed that the coefficient of variation of fluorescence readings was generally less than 5%, and the results of different batches of reagents were consistent.

[0026] Example 3, visual detection of lateral flow chromatographic test strips (LFA) After the fluorescence reaction of Example 2, 10 μL of reaction solution was taken from each tube and spotted on the sample pad of the nucleic acid test strip. The reaction mixture was allowed to move along the test strip by capillary action for 5 minutes. In positive samples, the FAM-labeled fragment (bound to gold nanoparticles) of the reporter probe cleaved by Cas12a could move along the capillary and be captured and enriched at the detection line (T line, pre-coated with anti-FAM antibody), presenting a red band visible to the naked eye. The intact probe that was not cleaved was retained at the control line (C line, coated with streptavidin) due to the presence of Biotin-FAM at both ends, so the negative control only showed color development at the C line and no signal at the T line. The use of the 4FAM-Biotin probe in this example greatly enhanced the naked-eye recognition of the test strip: the T line was clearly red in positive samples, and even weakly positive samples (with a lighter T line) could be distinguished, while negative samples only showed the control line without non-specific background bands. To compare the effect of single FAM probes, the traditional FAM-Biotin probe was applied to the same system, and the results showed no T line color development in LFA mode and significantly lower signal than the 4FAM probe in fluorescence mode. Thus, the 4-valent fluorescent reporter probe played a key role in the detection platform of the present application, allowing direct interpretation of H1N1 nucleic acid detection results by test strip without any instrument.

[0027] Example 4, Clinical sample detection and verification This example evaluated the detection performance of the detection platform of the present application on real clinical samples. A total of 50 clinical throat swab samples were collected, of which 35 were confirmed positive for H1N1 by hospital RT-qPCR and 15 were negative (samples were collected during an influenza season, and all operations followed ethical approval and informed consent of the subjects). Each sample was extracted with the QIAamp Viral RNA kit to extract total RNA, and eluted in 60 μL of RNase-Free water for standby. Then the extracted RNA stock solution was diluted 5-fold, and 2 μL of the diluted solution was used as the template for direct addition to the DF-Cas12a fluorescence reaction system described in Example 2 for detection. After 30 minutes of reaction, the fluorescence reading was recorded and the positive / negative results were determined according to the threshold value.

[0028] The detection found that: among the 35 RT-qPCR positive samples, 32 samples showed positive fluorescence signals, and 3 samples were not detected (reported as false negatives, and the actual RT-qPCR Ct values of the 3 samples were all > 36, and the viral load was extremely low); among the 15 RT-qPCR negative samples, 14 samples were not detected, and 1 sample showed a weak positive signal (false positive, and the patient was rechecked and found to be infected with RSV virus). According to this, the positive coincidence rate (PPA) of the DF-Cas12a detection method of the present application relative to RT-qPCR is 91.4%, the negative coincidence rate (NPA) is 93.3%, the total coincidence rate is 92.0%, and the Kappa value is 0.85. On the same sample, the sensitivity of the traditional virus antigen rapid detection kit is only about 50%, which shows that the present application greatly improves the detection reliability. The total time from extracting RNA to obtaining results for each sample is about 1 hour, and the CRISPR detection step is 30 minutes, which meets the requirements of WHO for on-site diagnostic tests (POCT) in terms of sensitivity, specificity and time. The above clinical blind test verifies the effectiveness of the H1N1 detection platform of the present application, and provides support for its feasibility in practical application.

[0029] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A CRISPR-Cas12a-based H1N1 virus ultrasensitive nucleic acid detection system, characterized in that, The system includes: (a) A Cas12a recognition system comprising a Cas12a protease and a specific crRNA; wherein the Cas12a protease has PAM recognition capability; and wherein the crRNA comprises a guide sequence and a stem-loop structure complementary to the target nucleic acid of the H1N1 virus. (b) An allosteric regulatory component comprising a pair of complementary oligonucleotide sequences; one of the oligonucleotides hybridizes with a portion of the crRNA sequence to form a double-stranded structure in the absence of a target nucleic acid, thereby inhibiting the enzymatic activity of the Cas12a protease; the oligonucleotides are all phosphorylated at the 5' end for recognizing the PAM sequence at the end of the complementary oligonucleotide sequence after Cas12a binding. (c) A free nucleic acid probe system comprising a bipartite-crRNA combination and a reporter probe; the bipartite-crRNA combination consists of a bipartite-crRNA handle fragment, a bipartite-crRNA spacer fragment, and an activator oligonucleotide. The bipartite-crRNA handle is an RNA or DNA oligonucleotide containing a portion of the hairpin structure of the crRNA. The bipartite-crRNA spacer is an oligonucleotide whose sequence is partially complementary to the H1N1 target sequence. The activator is a short oligonucleotide complementary to the bipartite-crRNA spacer. When the Cas12a protease is initially activated by the target nucleic acid, its conformation changes, and it competes with the bipartite-crRNA combination for binding, replacing the original binding of crRNA to Cas12a, thereby reactivating the enzyme activity of Cas12a and triggering a secondary cleavage amplification signal of the free reporter probe. The reporter probe is labeled with a detectable marker, which generates a detectable signal when the reporter probe is cleaved by the activated Cas12a protease.

2. The system according to claim 1, characterized in that: The crRNA specifically recognizes a conserved fragment of the M1 gene of the H1N1 virus, the sequence of which is shown in SEQ ID NO.

1.

3. The system according to claim 1, characterized in that: One sequence of the allosteric regulatory component is complementary to the crRNA sequence and is 9-15 bp in length. Its nucleotide sequence is shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. The system according to claim 1, characterized in that: The signal amplification component consists of the nucleotide sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

5. The system according to claim 1, characterized in that: The free reporter probe is an oligonucleotide probe of 5-10 nt in length, with two FAM fluorescent groups labeled at each end and a BHQ1 quencher group and a Biotin molecule labeled at the middle via a thymine site. Its sequence is as follows: Biotin-TTATGT(FAM)TGTATGT(FAM)TGTATT(BHQ1)TTAT(FAM)TTATG-FAM.

6. The system according to claim 1, characterized in that: The Cas12a protease is derived from... Eubacterium rectale ErCas12a protease.

7. A method for detecting H1N1 virus nucleic acid for non-diagnostic purposes, characterized in that, Using the detection system as described in any one of claims 1-6, the method comprises the following steps: (1) Mix the sample to be tested with the detection system; (2) Incubate under conditions suitable for the Cas12a protease reaction; (3) Detect the signal generated by the reporting system, wherein the presence of the signal indicates the presence of H1N1 virus target nucleic acid in the sample.

8. The detection method according to claim 7, characterized in that: The sample to be tested is a throat swab or nasal swab sample collected from the patient to be tested.

9. A kit for detecting H1N1 virus, characterized in that, The kit comprises the detection system as described in any one of claims 1-6.

10. The reagent kit according to claim 9, characterized in that: The kit also includes on-site nucleic acid extraction / lysis reagents for rapidly releasing viral nucleic acid from samples such as throat swabs and sputum; and a portable fluorescence signal detection device or a handheld blue light excitation source for real-time monitoring or visual observation of fluorescence signals. And a nucleic acid test strip reading device for interpreting results in side-flow chromatography mode.

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