A nucleic acid panel, kit and method for detecting influenza A H1N1 virus

Abstract

The application belongs to the technical field of biological detection, and relates to a nucleic acid group, a kit and a method for detecting influenza A H1N1 virus. The nucleic acid group comprises RPA amplification primer pairs, crRNA and ssDNA probes, the nucleotide sequences of the RPA amplification primer pairs correspond to SEQ ID NO. 3-4, and the nucleotide sequences of the crRNA and the ssDNA are respectively shown as SEQ ID NO. 6 and 7. Based on the RPA and CRISPR / Cas12a technologies, the application establishes a single-tube detection method for the influenza A H1N1 virus and an OTRRC method for the influenza A H1N1 virus, and the detection result can be visualized through a test strip. The method has the advantages of rapidness, high sensitivity and good specificity, and shows good applicability in sample detection, the detection result is consistent with the gold standard RT-qPCR, and the method shows the potential as an efficient POCT tool.

Description

Technical Field

[0001] This invention relates to a nucleic acid genome, reagent kit, and method for detecting H1N1 influenza virus, belonging to the field of biodetection technology. Background Technology

[0002] The H1N1 influenza virus is a single-stranded negative-sense RNA virus. Due to its highly variable genome, wide host range, and strong infectivity, it can sometimes cause large-scale seasonal global pandemics. Early symptoms of H1N1 influenza are highly similar to those of the common cold, but the disease progresses rapidly and carries a higher risk of complications. Early and accurate detection of H1N1 influenza virus infection is crucial for disease treatment and prognosis. Furthermore, the typical manifestations of acute respiratory infection (ARI) following H1N1 influenza virus infection are similar to those following other influenza viruses, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Currently, there is no rapid or specific screening method to differentiate between H1N1 and other influenza viruses, making it difficult for doctors to determine whether an infection is caused by H1N1 influenza virus. Therefore, there is an urgent need to develop a rapid, sensitive, and specific detection method to identify the H1N1 influenza virus in order to meet testing requirements.

[0003] Currently, traditional diagnostic methods for H1N1 influenza A virus still have shortcomings. Virus isolation and culture methods suffer from high operator skill barriers, long processing times, and high costs; immunological detection methods have low sensitivity, may be limited by the window period, and are susceptible to cross-reactivity; traditional molecular biological detection methods, such as real-time quantitative polymerase chain reaction (RT-qPCR), require sophisticated equipment and precise thermal cycling. In recent years, isothermal nucleic acid amplification (INA) technology has become the preferred solution for nucleic acid detection of many pathogens. This method does not require thermal cycling, can perform multiple detection methods, and has high sensitivity, demonstrating significant advantages in various application scenarios such as laboratory, field testing, and home self-testing. Among them, recombinase polymerase amplification (RPA) has become the preferred nucleic acid amplification platform due to its simplicity, high sensitivity and specificity, and high tolerance to PCR inhibitors. However, RPA is sensitive to contamination and prone to non-specific amplification, leading to false positive results, which limits its application in high-specificity detection scenarios. CRISPR-associated (Cas) protein (CRISPR / Cas) technology, when applied to nucleic acid detection, generates fluorescent detection signals by cleaving single-stranded DNA (ssDNA) reporter genes labeled with fluorescent and quenching groups. Cas12a, guided by crRNA, specifically recognizes target DNA, cleaves the target DNA upon activation, exhibits trans-cleavage activity, and indiscriminately cleaves surrounding single-stranded DNA, amplifying the signal to produce strong fluorescence. However, in CRISPR / Cas12a detection systems, the design of the ssDNA reporter probe is one of the key factors determining the system's sensitivity and specificity. Current technologies face the following unresolved technical challenges in the design and optimization of this probe: 1. The contradiction between structural stability and cleavage efficiency: ssDNA probes need to maintain a single-stranded linear structure to avoid secondary structures interfering with the trans-cleavage activity of Cas12a. However, current technologies struggle to balance probe thermal stability and enzymatic cleavage efficiency without forming hairpins or self-complementary structures. 2. Probe length typically needs to be between 18–30 nt. Too short a length leads to easy degradation or unstable binding, while too long a length reduces cleavage efficiency. However, current technologies lack a basis for optimizing probe length for specific pathogens (such as H1N1). 3. Non-specific interference is difficult to avoid. The probe sequence must avoid homologous sequences with the target DNA, crRNA, or other nucleic acid components in the system; otherwise, non-specific hybridization or increased background signal may occur. However, current design methods cannot completely avoid potential homologous regions.

[0004] Due to the aforementioned multiple limitations, existing ssDNA probes have low design success rates and poor reproducibility. If poorly designed, they can easily lead to false negatives or a significant decrease in sensitivity, severely limiting the reliability of the CRISPR / Cas12a system in samples. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the aforementioned problems in existing technologies, this invention provides a nucleic acid genome, kit, and method for detecting influenza A (H1N1) virus. By systematically optimizing the sequence length, base composition, GC content, and secondary structure prediction of the ssDNA probe, this invention provides, for the first time, a highly sensitive, low-background, and structurally stable ssDNA reporter probe suitable for detecting influenza A (H1N1) virus, solving the problems of difficult probe design and unstable performance in existing technologies.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a nucleic acid genome for detecting influenza A (H1N1) virus, comprising an RPA amplification primer pair, crRNA, and an ssDNA probe, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of the crRNA is shown in SEQ ID NO.6, and the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.7.

[0010] The nucleotide sequence described above also includes a positive control template nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO.1. This positive control template nucleic acid molecule contains the binding site for the RPA amplification primer pair and the recognition site for crRNA. The positive control is used to verify the effectiveness of the RT-RPA amplification and CRISPR / Cas12a cleavage system. It reacts synchronously with the sample during detection and requires a positive signal (fluorescence reaching the target or color development of the T line on the test strip); otherwise, the experimental results are invalid.

[0011] The positive control template nucleic acid molecule can be RNA, corresponding to a specific conserved region of the H1N1 influenza A virus RNA, or a synthetically produced nucleic acid fragment containing the core target sequence of the viral RNA. The positive control template nucleic acid molecule must contain the binding sites of the RPA primer pairs (SEQ ID NO. 3, 4) and the recognition site of crRNA (SEQ ID NO. 6) to be specifically amplified and recognized by the core nucleic acid group, simulating the detection process of real viral RNA. Alternatively, the positive control template nucleic acid molecule can be a cDNA fragment reverse transcribed from viral RNA. It is not a "direct comparison" with the viral RNA, but rather added to the detection system as a "positive reference template." If the system produces a specific fluorescent signal for this positive control (i.e., the detection is effective), it proves that the RPA amplification, CRISPR / Cas12a cleavage, and other processes are all normal; if the test sample (containing viral RNA) also produces a fluorescent signal, the sample is confirmed as positive; otherwise, it is negative.

[0012] In the aforementioned nucleic acid genome, preferably, the ssDNA probe is labeled with a fluorescent group and a quencher group at both ends.

[0013] Furthermore, the fluorescent group is any one of FAM, HEX, Red, Cy3, Cy5, Cy7, FITC, JOE, TET, VIC, etc., and the quenching group is any one of BHQ1, TMARA, BHQ2, BHQ3, MGB, etc.

[0014] Secondly, the present invention also provides a kit for detecting influenza A (H1N1) virus, which includes the nucleic acid genome as described above.

[0015] The kit described above also includes a lateral immunochromatographic test strip, wherein the sample pad of the lateral immunochromatographic test strip is coated with a colloidal gold-labeled anti-FAM monoclonal antibody, the detection line is coated with goat anti-mouse secondary antibody, and the control line is coated with avidin.

[0016] The lateral immunochromatographic test strip determines whether the Cas12a enzyme is activated by the presence or absence of the test line, and determines the validity of the test strip by the presence or absence of the control line. The specific criteria are as follows: if both the control line and the test line are colored, the sample is positive; if the control line is colored but the test line is not colored, the sample is negative; if the control line is not colored, the test strip is invalid and the test result is invalid.

[0017] In the detection kit described above, preferably, the two ends of the ssDNA probe are labeled with the fluorescent groups FAM and Biotin, respectively.

[0018] It should be noted that when using test strips, the two ends of the ssDNA probe are labeled with FAM and Biotin, respectively.

[0019] When using real-time PCR, the two ends of the ssDNA probe are labeled with FAM and BHQ1, respectively.

[0020] The detection kit described above, preferably, further includes RPA lyophilized powder, rehydration buffer, DEPC-treated water, MgOAc, reverse transcriptase, RNaseH, DTT, and Cas12a protein; the RPA lyophilized powder contains recombinase, single-stranded binding protein SSB, and DNA polymerase.

[0021] The application of nucleic acid omics in the preparation of visual detection reagents, as described above.

[0022] Thirdly, the present invention provides a visualization reagent for detecting influenza A (H1N1) virus, comprising a detection reagent and a display reagent. The detection reagent comprises a bottom reagent placed at the bottom of a reaction tube and a cap reagent placed inside the cap of the same reaction tube. The bottom reagent comprises an RPA amplification system of primer pairs with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4. The cap reagent comprises a CRISPR / Cas12a cleavage system of crRNA with nucleotide sequence as shown in SEQ ID NO.6, an ssDNA probe with nucleotide sequence as shown in SEQ ID NO.7, Cas12a protein, and buffer, wherein the ssDNA probe is modified with fluorescent groups X and Biotin at both ends, respectively.

[0023] The display reagent is a lateral immunochromatographic test strip. The sample pad of the lateral immunochromatographic test strip is coated with a colloidal gold-labeled monoclonal antibody against fluorescent group X, the detection line is coated with goat anti-mouse secondary antibody, and the control line is coated with avidin.

[0024] The lateral immunochromatographic test strip determines whether the Cas12a enzyme is activated by the presence or absence of color development on the detection line.

[0025] The fluorescent group X is any one of FAM, HEX, Red, Cy3, Cy5, Cy7, FITC, JOE, TET, VIC, etc.

[0026] The visualization reagents shown above contain rehydration buffer, DEPC-treated water, forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, half-gravel RPA lyophilized, MgOAc, reverse transcriptase, RNaseH, and DTT;

[0027] The capping reagent includes Cas12a, NEBuffer™ r2.1, the crRNA, and the ssDNA.

[0028] Furthermore, the visualization reagent described above comprises 14.75 μL of rehydration buffer, 5.75 μL of DEPC-treated water, 1.00 μL each of forward and reverse primers (both at a concentration of 480 nM), half a lyophilized RPA particle, 1.25 μL of MgOAc, 0.24 μL of 200 U / μL reverse transcriptase, 0.48 μL of 5 U / μL RNase H, and 0.5 μL of DTT;

[0029] The capping reagent comprises 1.6 μL DEPC-treated water, 3.5 μL 100 nM Cas12a (Cpf1), 3.5 μL 10×NEBuffer™ r 2.1, 0.35 μL 10 μM crRNA, and 1.05 μL 300 nM ssDNA.

[0030] Fourthly, the present invention provides a method for detecting influenza A (H1N1) virus, which is a non-diagnostic detection method, comprising the following steps:

[0031] S1. Extract viral RNA from the sample to be tested;

[0032] S2. Take the viral RNA extracted in S1 and add it to the RT-RPA amplification system containing primer pairs with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, which are placed at the bottom of the reaction tube. Amplify at a constant temperature and obtain the RPA amplification product at the bottom of the tube.

[0033] S3. By momentary centrifugation, the crRNA containing the nucleotide sequence shown in SEQ ID NO.6, the ssDNA probe containing the nucleotide sequence shown in SEQ ID NO.7, the Cas12a protein, and the buffer CRISPR / Cas12a cleavage system, which were placed inside the cap of the reaction tube beforehand, are transferred to the bottom of the tube and mixed with the RPA amplification product, and then incubated.

[0034] S4. Result Judgment: The result shall be judged using one of the following two methods:

[0035] Method 1: In step S3, the incubation process is carried out in a fluorescence PCR instrument, and the detection results are judged by real-time acquisition of fluorescence signals; at this time, the two ends of the ssDNA probe are modified with fluorescent groups and quenching groups, respectively.

[0036] Method 2: After the incubation in step S3 is completed, add the incubated product to a lateral immunochromatographic test strip for detection.

[0037] At this time, the two ends of the ssDNA probe are modified with fluorescent group X and Biotin, respectively; the sample pad of the lateral immunochromatographic test strip is coated with a colloidal gold-labeled monoclonal antibody against fluorescent group X, the detection line is coated with goat anti-mouse secondary antibody, and the control line is coated with avidin.

[0038] The criteria for judgment are as follows: if both the control line and the test line show color, the result is positive; if the control line shows color but the test line does not, the result is negative; if neither the test line nor the control line shows color, the test strip is considered invalid and needs to be tested again.

[0039] In the method described above, preferably, in step S1, viral RNA extraction uses a nucleic acid extraction-free release agent.

[0040] Furthermore, the nucleic acid extraction-free release agent is QuickExtract DNA Extraction Solution.

[0041] In the method described above, preferably, in step S2, the isothermal amplification is performed at a temperature of 37℃±1℃ for 10~15 min; in step S3, when using a fluorescence PCR instrument for detection, the incubation temperature is 37℃±1℃ for 30 min; when using a lateral immunochromatographic test strip for detection, the incubation temperature is 37℃±1℃ for 10 min.

[0042] Preferably, the RT-RPA system described above comprises 14.75 μL rehydration buffer, 5.75 μL DEPC-treated water, 1.00 μL each of forward and reverse primers (both at a concentration of 480 nM), half a lyophilized RPA particle, 1.25 μL MgOAc, 0.24 μL 200 U / μL reverse transcriptase, 0.48 μL 5 U / μL RNase H, and 0.5 μL DTT;

[0043] The CRISPR / Cas12a system comprises 1.6 μL DEPC-treated water, 3.5 μL 100 nM Cas12a, 3.5 μL 10×NEBuffer™ r2.1, 0.35 μL 10 μM crRNA, and 1.05 μL 300 nM ssDNA.

[0044] As described above, nuclease-free water was used as a negative control, and a plasmid containing the sequence shown in SEQ ID NO.1 was used as a positive control for detection. During fluorescence PCR detection, the average (F-NTC_mean) and standard deviation (SD-NTC) of the fluorescence intensity obtained from ≥8 negative controls (NTC) during the 10-minute fluorescence stabilization phase were calculated, and F-NTC_mean + 3 × SD-NTC was used as the judgment threshold. When interpreting samples, if the fluorescence value of the sample reaches or exceeds the threshold at any time point within 30 minutes, and the fluorescence curve shows a continuous upward trend, it is judged as positive. If the fluorescence signal is always below the threshold and the NTC signal is stable throughout the reaction, it is judged as negative. If the sample fluorescence is between F-NTC_mean + 2 × SD-NTC and the judgment threshold, or the signal is unstable, the curve does not show a continuous upward trend, or some controls show abnormalities, it is judged as suspicious and retesting is recommended.

[0045] (III) Beneficial Effects

[0046] The beneficial effects of this invention are:

[0047] This invention provides a nucleotide genome for detecting H1N1 influenza A virus. Designed primers enable efficient, specific, and rapid amplification of H1N1 influenza A virus to obtain RPA amplification products. Corresponding crRNAs are designed for specific regions of the RPA amplification products. Only when the RPA amplification product is a specific target nucleic acid can Cas12a bind to the target nucleic acid under the guidance of crRNA and undergo a conformational change, thereby being activated. If the RPA amplification product is a non-specific amplification product, its sequence does not match the crRNA, and the Cas12a cleavage activity cannot be activated. The crRNA design effectively eliminates non-specific RPA amplification products, avoiding false positives. The ssDNA probe, as a reporter gene, is the core of the detection signal output; successful design of the ssDNA probe effectively avoids false negatives. This nucleotide genome is used for real-time fluorescence detection and lateral immunochromatographic strip detection.

[0048] This invention establishes a one-tube assay based on RT-RPA and CRISPR / Cas12a (OTRRC method) for the detection of H1N1 influenza A virus. Using real-time fluorescence detection and lateral flow immunoassay strips as detection methods, it is termed OTRRC-FD (OTRRC fluorescence detection) and OTRRC-LFIA (OTRRC lateral flow immunoassay). Using H1N1 influenza A virus plasmids and pseudoviruses as templates, the sensitivity and specificity of the OTRRC-FD and OTRRC-LFIA methods were analyzed. Results show that the OTRRC-FD method can detect as low as 2.8 × 10⁻⁶ viruses within 20 minutes. -1 The standard plasmid can detect as few as 1.71 × 100 copies / μL of pseudovirus in 30 minutes; the OTRRC-FD method can detect as few as 10 copies / μL of standard plasmid template in 30 minutes and as few as 101 copies / μL of pseudovirus in 35 minutes, and there is no cross-reactivity with SARS-CoV-2, IBV-Victoria lineage, and RSV.

[0049] The methods for detecting H1N1 influenza A virus provided by this invention include fluorescence detection and test strip detection, with detection sensitivities of 95% and 90% for real samples, respectively, specificity of 100% for both, and consistency with RT-qPCR of 97.19% and 94.56%, respectively.

[0050] The visualization reagent for detecting H1N1 influenza A virus provided by this invention visualizes the test results through test strips, allowing for visual judgment of the results without the need for special testing instruments. This method has the advantages of being rapid (within 35 minutes), highly sensitive, and having good specificity, and it shows good applicability in sample testing. The test results are in good agreement with the gold standard RT-qPCR, demonstrating its potential as an efficient point-of-care testing tool. Attached Figure Description

[0051] Figure 1 This is a schematic diagram illustrating the detection principle and process of the present invention;

[0052] Figure 2 These are the primer screening results;

[0053] Figure 3 Results of RPA primer concentration optimization;

[0054] Figure 4 The results are for optimizing the reaction temperature;

[0055] Figure 5Results of the optimized Cas12a to crRNA ratio;

[0056] Figure 6 Results of ssDNA fluorescent probe concentration optimization;

[0057] Figure 7 Optimization results for Cas12a concentration;

[0058] Figure 8 Results of RPA amplification time optimization;

[0059] Figure 9 Sensitivity analysis of OTRRC-FD method using plasmid as template;

[0060] Figure 10 Sensitivity analysis of the OTRRC-LFIA method using plasmid as a template;

[0061] Figure 11 Sensitivity analysis of the OTRRC-FD method using pseudoviruses as templates;

[0062] Figure 12 For the specificity analysis of the OTRRC-FD method using pseudoviruses as templates;

[0063] Figure 13 Sensitivity and specificity analysis of the OTRRC-LFIA method using pseudoviruses as templates;

[0064] Figure 14 This is a comparison chart of sample detection results using the OTRRC method and the RT-qPCR method. Detailed Implementation

[0065] This invention establishes a one-tube assay based on RT-RPA and CRISPR / Cas12a (OTRRC) for the detection of influenza A H1N1 virus by combining reverse transcription RPA (RT-RPA) and CRISPR / Cas12a. This enables rapid and efficient detection of influenza A H1N1 virus while effectively reducing the risk of contamination. Furthermore, to expand the detection scenarios, this invention combines the OTRRC method with a lateral flow immunoassay (LFIA) platform. The signal molecules cleaved by CRISPR pass through LFIA, undergo immunochromatography, colloidal gold aggregation and color development, and result interpretation, allowing for naked-eye interpretation of the test results. This establishes a more convenient and rapid point-of-care testing (POCT) method.

[0066] The OTRRC detection process and principle established in this invention are as follows: Figure 1As shown in the diagram, firstly, an extraction solution is added to the sample to be tested and incubated at 95°C for 5 min. This allows the viral particles to rapidly lyse and fully release RNA under the combined action of high temperature and chemical lysis agents, eliminating the need for traditional nucleic acid extraction and improving overall detection speed and ease of operation. Subsequently, 5 μL of the released viral RNA is added to the RT-RPA reaction system and amplified at 37°C for 10 min. The RT-RPA system comprises reverse transcriptase, recombinase, primers, single-stranded binding protein (SSB), and DNA polymerase in specific proportions, forming a synergistic reaction system: viral RNA is preferentially and rapidly converted to cDNA by reverse transcriptase; the recombinase binds to the primers to form a primer-protein complex, which can recognize the target sequence and initiate the strand displacement reaction under isothermal conditions at 37°C; the displaced single-stranded DNA specifically binds to SSB, preventing strand re-annealing and improving amplification efficiency; and the DNA polymerase performs strand amplification at the primer extension end. This multi-component combination provides the ability to achieve exponential amplification under low-temperature isothermal conditions and is a non-obvious synergistic system, providing sufficient target DNA template for subsequent CRISPR detection.

[0067] To avoid aerosol contamination caused by tube opening, this invention pre-places the CRISPR / Cas12a system inside the reaction tube cap and mixes it with the RT-RPA amplification product via a transient separation method, enabling continuous amplification and detection within a single tube. After mixing, the mixture is incubated at 37°C for 10 min. The CRISPR / Cas12a-crRNA complex specifically recognizes the PAM sequence on the RT-RPA amplification product. The crRNA pairs complementaryly with the target DNA, activating Cas12a. The activated Cas12a exhibits strong trans-cleavage activity, enabling non-specific cleavage of the ssDNA fluorescent probe in the system, releasing quenched fluorescent groups and generating a directly detectable fluorescent signal. Through the synergistic reaction of the enzyme system and the single-tube closed operation process, this invention not only achieves rapid detection with high sensitivity, high specificity, and low contamination risk, but also ensures that the entire detection process is completed within approximately 30 min, significantly improving the feasibility of on-site detection and POCT applications. This invention achieves signal output using a qPCR instrument (single-tube fluorescence method) or a lateral flow immunoassay strip (single-tube strip method), which are respectively called OTRRC-FD (OTRRC fluorescence detection) and OTRRC-LFIA (OTRRC lateral flow immunoassay) methods to detect the nucleic acid of influenza H1N1 virus.

[0068] The OTRRC-LFIA test strip works as follows: The lateral immunochromatographic strip is placed in the reaction tube containing the test mixture. The reaction system migrates under capillary action. Intact reporter molecules (labeled with Biotin and FAM at both ends) allow the colloidal gold to be completely captured at the control line. When a reporter molecule is cleaved by Cas enzyme, the colloidal gold bound to the cleaved fragment cannot be captured by the control line, forming the test line. The presence or absence of the test line indicates whether Cas enzyme has been activated. The reporter molecule ssDNA probe used is labeled with FAM (5' end) and Biotin (3' end) at both ends, respectively. Without a target: Cas12a is not activated, the ssDNA probe is intact, and Biotin binds to avidin on the control line (C line), resulting in only C line color development (indicating a negative result). With a target: Cas12a is activated and cleaves the ssDNA probe, releasing the FAM-labeled short chain. This short chain binds to the colloidal gold-labeled anti-FAM antibody and migrates to the detection line (T line), where it is captured by goat anti-mouse secondary antibody, resulting in visible color development on the T line (indicating a positive result).

[0069] Secondly, regarding primer design, existing RPA primers exhibit significant sequence bias, easily leading to dimer formation or mismatches with slight inappropriate selection. Based on extensive comparisons of H1N1 influenza virus gene sequences, this invention significantly reduces the probability of non-specific binding and improves amplification efficiency and success rate by screening conserved regions, limiting GC content (40%–55%), avoiding more than 6 consecutive bases, and strictly controlling primer length to 30–35 nt. These design parameters are not empirical values ​​conventionally used in existing RPA literature, but rather an optimized model established for the H1N1 gene structure and background sequence complexity, demonstrating the non-obviousness of this invention's primer selection strategy.

[0070] In the design of ssDNA reporter probes, existing technologies do not provide clear sequence and structural guidance. This invention, by comparing ssDNA of different lengths and base compositions and combining the trans-cleavage kinetics of Cas12a, determines that the ssDNA should be controlled between 8-24 nt; avoid more than 6 consecutive identical base repeats; limit the GC content to 30%-50%; and strictly remove self-complementary regions that may form hairpin structures. It also ensures that Cas12a can quickly recognize and cleave the probe after activation, avoiding signal loss due to probe failure, thus obtaining a reporter probe with optimal cleavage efficiency and lowest background. The combination of the fluorophore and quencher groups of the ssDNA probe has been optimized and is ultimately preferred to be FAM-BHQ1, with an initial quenching efficiency of over 95% and high fluorescence release efficiency after cleavage. Even with a small amount of Cas12a activated by a low-abundance target, a recognizable fluorescent signal can be generated, reducing false negatives due to the presence of the target but insufficient signal.

[0071] This invention, through extensive experimental research, has found that using the conventional two-tube method—that is, placing 25 μL of RPA amplification system in one PCR tube and incubating at 37°C for 15 min to complete amplification; after amplification, opening the tube cap, using a pipette to aspirate 5 μL of RPA product, adding it to 10 μL of CRISPR / Cas12a system pre-alimited in another PCR tube cap, mixing well, incubating at 37°C for 10 min, and then detecting on a fluorescence PCR instrument for 30 min, collecting fluorescence signals every 30 s—significantly increases the false positive rate compared to the single-tube instantaneous mixing method, and lowers the limit of detection to 10. 2 -10 3 The copies / µL ratio is significantly lower than the 2.8 × 10⁻⁶ for single-tube detection in this invention. -1 copies / µL (20 min) and 1.7 copies / µL (30 min).

[0072] When the RT-RPA amplification temperature is adjusted to 25℃ (low temperature) or 42℃ (high temperature), while the CRISPR step remains at 37℃, the RPA amplification efficiency decreases significantly, resulting in insufficient CRISPR cleavage and failing to achieve the sensitivity of this invention. Therefore, the preferred RPA amplification temperature is 37℃ ± 1℃. When the MgOAc concentration in the RT-RPA amplification system is adjusted to 1mM (too low) or 14mM (too high), phenomena such as no amplification or increased false positives occur, indicating that the MgOAc concentration specified in this invention is insufficient. 2+ The preferred concentration is 3~7 mM, and the most preferred concentration is 3.57 mM.

[0073] When the concentration of Cas12a in the CRISPR / Cas12a system is too low (0.1×) or too high (3×), the detection sensitivity decreases by 1–2 logarithmic levels, and the rapid detection capability obtained by this invention cannot be achieved. Therefore, the preferred concentration and amount of Cas12a is 3.5 μL 1000 nM, that is, the preferred final concentration is 100 nM.

[0074] To better explain and facilitate understanding of this invention, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, conventional techniques in the art are employed, and commercially available reagents are used.

[0075] It should be noted that in all experiments of this invention, each group was performed three times independently. The mean of the fluorescence signal at the stable fluorescence intensity (10 min) ± 3 times the standard deviation is used, and the error bar represents the standard deviation of the three replicate experiments. The mean (F-NTC_mean) and standard deviation (SD-NTC) of the fluorescence intensity obtained from ≥8 negative controls (NTC) at the 10 min fluorescence stabilization stage were calculated, and F-NTC_mean + 3 × SD-NTC was used as the judgment threshold. When interpreting samples, if the fluorescence value of the sample reaches or exceeds the threshold at any time point within 30 min, and the fluorescence curve shows a continuous upward trend, it is judged as positive; if the fluorescence signal is always below the threshold and the NTC signal is stable throughout the reaction, it is judged as negative. If the sample fluorescence is between F-NTC_mean + 2 × SD-NTC and the judgment threshold, or the signal is unstable, the curve does not show a continuous upward trend, or some controls show abnormalities, it is judged as suspicious and retesting is recommended. If the positive control in this batch is not positive, or the NTC shows an abnormal increase, it is judged as an invalid result and retesting is required. Data processing and statistical analysis were performed using SPSS 25.0. t-tests and one-way ANOVA were used to analyze the statistical significance of differences between the experimental groups and the negative control group. Paired chi-square tests or Fisher's exact test were used to analyze whether there was a difference in detection rate between the detection method and the "gold standard" RT-qPCR. P < 0.05 was considered statistically significant. Origin 2021 was used for plotting.

[0076] Example 1

[0077] This embodiment provides a nucleic acid genome and kit for detecting human influenza A (H1N1) virus. The design and screening methods for the nucleic acid genome are as follows:

[0078] 1. Materials and Methods

[0079] 1.1 Sources of Samples and Virus Standards

[0080] Twenty human H1N1 influenza A virus, five novel coronavirus (SARS-CoV-2), five influenza B virus Victoria lineage, and five respiratory syncytial virus RNA samples were obtained from the Chengdu Center for Disease Control and Prevention; inactivated H1N1 influenza A virus, novel coronavirus, influenza B virus Victoria lineage, and respiratory syncytial virus reference materials were purchased from Henan Wanjia Reference Material R&D Center.

[0081] 1.2 Reagents and Consumables

[0082] Standard plasmids, primers, ssDNA fluorescent probes, and crRNA were synthesized by Shanghai Sangon Biotech Co., Ltd. The QuickExtract DNA Extraction Solution was purchased from LGC Biosearch Technologies (USA). The RPA kit was purchased from TwistDX™ Ltd. (UK), SuperScript™ IV reverse transcriptase and DEPC-treated water were purchased from Thermo Fisher Scientific (USA), and RNase H enzyme, EnGen® Lba Cas12a (Cpf1) nuclease, and NEBuffer™ r2.1 (10×) were purchased from New England Biolabs (USA). CRISPR nucleic acid detection test strips were purchased from Shenzhen Yizhi Biotechnology Co., Ltd.

[0083] 1.3 Establishment of OTRRC Method using RT-RPA-CRISPR / Cas12a Single-Pipe Method

[0084] 1.3.1 Target gene screening, RPA primer design and selection

[0085] The IAV-H1N1 M gene nucleotide sequence was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and compared to obtain the target gene nucleotide sequence as shown in SEQ ID NO.1. A standard plasmid was designed based on this sequence. Primers for the H1N1 influenza A virus were designed using Primer Premier V5.0 software. A large number of primers were screened to select the most sensitive and specific primer sets. To save space, only three pairs of RPA forward and reverse candidate primers are shown as examples. The specific sequences are shown in Table 1.

[0086] Table 1: Standard plasmids and primers for conserved genes of influenza A (H1N1) virus

[0087]

[0088] Note: F1 / 3 represents forward primer 1 and forward primer 3, F2 represents forward primer 2; R1 / 2 represents reverse primer 1 and reverse primer 2, R3 represents reverse primer 3.

[0089] 1.3.2 RPA Primer Screening

[0090] Screening for the optimal primer pair for RPA of H1N1 influenza A virus, with a concentration of 2.8 × 10⁻⁶. 5Using the standard plasmid (copies / μL) as a template, the amplification system was prepared according to the RPA Basic kit (TwistDX, catalog number TABAS03KIT): One RPA microsphere powder was added to 29.5 μL of rehydration buffer, and gently mixed to form the RPA reaction base solution. 2.4 μL of forward and reverse primers (see Table 1) and 10.7 μL of DEPC-treated water were added, and after thorough mixing and centrifugation, the mixture was divided into two 22.5 μL tubes (as the test group and negative control group). For the positive control group, 1.25 μL of gene plasmid (sequence shown in SEQ ID NO. 1 in Table 1, concentration 2.8 × 10⁻⁶) was added. 5 The negative control group used an equal volume of DEPC-treated water instead of the standard plasmid (denoted as NTC). The reaction system was thoroughly mixed, briefly centrifuged, and then incubated at 39°C for 20 min in a dry incubator. After amplification, the RPA amplification product was purified (by adding an equal volume of phenol / chloroform (1:1) extraction buffer), thoroughly mixed, centrifuged, and the supernatant was collected. 5 μL of 6×SuperStain Loading Buffer was added at a 1:5 ratio, and 5 μL of the sample was loaded. Electrophoresis was performed at 110V for 30 min, and images were acquired using a gel imaging analyzer.

[0091] RPA amplification primer screening electrophoresis results are as follows: Figure 2 As shown in the figure, lane 1 is the 20bp DNA ladder; lanes 2 and 3 are the NTC and amplification groups using primer F1R1; lanes 4 and 5 are the NTC and amplification groups using primer F2R2; and lanes 6 and 7 are the NTC and amplification groups using primer F3R3. All three candidate RPA primer pairs (F1R1, F2R2, and F3R3) successfully amplified the target bands (lanes 3, 5, and 7) matching the expected target fragment size for the H1N1 influenza A virus. The negative control group using candidate primer F2R2 showed no nonspecific amplification bands, while the other two groups showed nonspecific amplification bands. Therefore, the primer pair with the F2 sequence shown in SEQ ID NO. 3 and the R2 sequence shown in SEQ ID NO. 4 was selected as the primers for the RPA amplification reaction.

[0092] 1.3.3 Design and Synthesis of crRNA and ssDNA Probes

[0093] Based on the optimal primer set F2 and R2 selected through RPA primer screening, the target fragment sequence was determined. Following the CRISPR / Cas12a crRNA design principles, crRNA and single-stranded DNA (ssDNA) probes with fluorescent and quenching groups modified at the 5' and 3' ends, respectively, were designed using the CHOPCHOP website (http: / / chopchop.cbu.uib.no / ). The ssDNA was labeled with FAM at the 5' end and BHQ1 at the 3' end, as shown in Table 2. By comparing the experimental performance of different candidate crRNA and ssDNA probes in terms of cleavage efficiency, fluorescence signal-to-noise ratio, and background level, this invention ultimately screened the optimal crRNA and ssDNA combination with the fastest reaction speed, strongest signal, and lowest background. The optimized crRNA and ssDNA probes are shown in Table 2.

[0094] Table 2 crRNA and ssDNA probes

[0095]

[0096] 1.3.4 Establishment of the detection procedure

[0097] The standard detection procedure for the OTRRC method is as follows: The 35 μL detection system consists of two parts: Part A (bottom of the tube) and Part B (inside the cap). Part A is a 25 μL RPA amplification system, including 1.00 μL each of forward and reverse primers F2 and R2, half a grain of lyophilized RPA powder (from the TwistAmp® Basic kit), 1.25 μL plasmid DNA, 14.75 μL rehydration buffer, 5.75 μL DEPC-treated water, and 1.25 μL MgOAc. Part B is a 10 μL CRISPR / Cas12a digestion system, including 3.5 μL 1μM EnGen® Lba Cas12a (Cpf1), 3.5 μL 10×NEBuffer™ r2.1, 0.35 μL 10 μM crRNA, 1.05 μL 10 μM ssDNA, and 5.1 μL DEPC-treated water. Part A is added to the bottom of the reaction tube, and Part B is pre-packaged inside the cap of the same reaction tube.

[0098] Incubate at 37℃ for 10 min to complete RPA amplification. Centrifuge the B part (CRISPR / Cas12a cutting system) inside the tube cap to the bottom of the tube and mix it thoroughly with the A part (RPA amplification product). Place the tube on a qPCR instrument and react at 37℃ for 30 min. Collect fluorescence signals every 30 s.

[0099] It should be noted that when testing the sample, the plasmid DNA in the 25 μL RPA amplification system in Part A should be replaced with the RNA of the sample to be tested, and 0.24 μL SuperScript™ IV reverse transcriptase, 0.48 μL RNase H, and 0.5 μL 100 mM DTT (bis(p-chlorophenyl)trichloroethane) should be added to Part A. The final reaction concentration of DTT is 5 mM, and the remaining components are consistent with the standard reaction system described above.

[0100] 1.3.5 Condition Optimization

[0101] Under standard detection procedures, primer concentration, CRISPR / Cas12a system, reaction temperature, and RPA amplification time were optimized according to the principle of controlling a single variable. For primer concentration optimization, four sets of primers with different concentrations were set up (each set included a negative control group), and the experiment was repeated three times, measuring the fluorescence signal intensity of each set. For the CRISPR / Cas12a system optimization, different Cas12a to crRNA ratios (1:1, 1:2, 2:1), different Cas12a concentrations (30, 50, 70, 100 nM), and different ssDNA probe concentrations (100, 200, 300 nM) were set up (each set included a negative control group), and the experiment was repeated three times, measuring the fluorescence signal intensity of each set. The RPA reaction temperature is 37–42℃; therefore, three different reaction temperatures of 37℃, 39℃, and 42℃ were optimized (each set included a negative control group), and the experiment was repeated three times, measuring the fluorescence signal intensity of each set. The RPA amplification time was optimized to 0, 2, 4, 6, 8, 10, and 12 min, with 7 groups of RPA amplification time conditions (each group had a negative control group). The experiment was repeated three times, and the fluorescence signal intensity of each group was measured.

[0102] The results of conditional optimization are as follows Figures 3-8 As shown in the figure, "***" indicates P < 0.001, "**" indicates P < 0.001, and "*" indicates P < 0.05, indicating statistical significance; "ns" indicates P > 0.05, indicating no statistical significance.

[0103] The negative control (NTC) showed the lowest fluorescence intensity, and the optimal conditions were selected based on cost-effectiveness. The results showed that an RPA primer concentration of 480 nM was optimal. Figure 3 (As shown); the optimal reaction temperature of the system is 37℃ ( Figure 4 As shown); the optimal ratio of Cas12a to crRNA is 2:1. Figure 5 As shown); the optimal concentration of the ssDNA fluorescent probe is 300 nM ( Figure 6 As shown); the optimal concentration of Cas12a is 100 nM (as shown). Figure 7As shown); the fluorescence value increases to a plateau phase after about 10 minutes of amplification, and 10 minutes is the time when the RPA amplification efficiency is optimal (as shown). Figure 8 (As shown).

[0104] 1.3.6 Preparation and Result Interpretation of the OTRRC-LFIA Reaction System

[0105] The lateral immunochromatographic test strips used in this invention can be prepared in-house or purchased from Shenzhen Yizhi Biotechnology Co., Ltd. Specifically, the sample binding pad of the lateral immunochromatographic test strip is pre-coated with colloidal gold-labeled mouse anti-FAM monoclonal antibody; the detection line of the nitrocellulose membrane is coated with goat anti-mouse secondary antibody to capture the free colloidal gold-mouse anti-FAM monoclonal antibody-FAM complex after cleavage; the control line is coated with avidin to capture the intact reporter probe (5'-FAM-ssDNA-3'-Biotin), with FAM and Biotin labeled at both ends. The intact reporter probe allows all the colloidal gold to be captured at the control line. When a reporter molecule is cleaved by Cas enzyme, the colloidal gold bound to the cleavage fragment cannot be captured by the control line, forming the detection line. The presence or absence of color development at the detection line indicates whether the Cas enzyme is activated, thus indicating the presence of the target gene in the analyte. The RT-RPA-CRISPR / Cas12a system prepared according to 1.3.4 (note that the two ends of the ssDNA probe are labeled with FAM and Biotin, respectively) is amplified and digested at 37℃. After dilution, the sample is inserted into the test strip and the result can be read after 5 minutes: if the test line is colored, it is considered positive, indicating that the sample contains the target gene; if the test line is not colored, it is considered negative, indicating that the target gene is not present. If neither the control line nor the test line is colored, it indicates an operational error or that the test strip has deteriorated and become ineffective. The test result is invalid, and a new test strip should be used or the test should be repeated.

[0106] Prepare a 35 μL RT-RPA-CRISPR / Cas12a detection system according to 1.3.4, with the difference being that the ssDNA probe is labeled with FAM and Biotin at both ends, respectively. Incubate at 37°C for 15 min to achieve RPA amplification. Then, briefly centrifuge and thoroughly mix the RPA amplification product with CRISPR / Cas12a, and incubate at 37°C for 10 min using a dry incubator. After the reaction is complete, add 25 μL of DEPC-treated water to dilute the system. Insert the lateral immunochromatographic test strip with the sample pad end facing down (the liquid level should not exceed the Max line), and leave it at room temperature for 5 min. Remove the strip and place it flat on the table to interpret the results. Negative: Control line shows a band, test line shows no band. Positive: Both test line and control line show bands. Invalid: Control line shows no band.

[0107] Example 2

[0108] This embodiment verifies the sensitivity of the human H1N1 influenza A detection kit provided in Example 1. The method is as follows:

[0109] 1. Sensitivity detection using plasmids as templates

[0110] With a concentration of 2.8 × 10 4 2.8×10 3 2.8×10 2 2.8×10 1 2.8×10 0 2.8×10 -1 Using copies / μL of the M gene plasmid (sequence shown in SEQ ID NO.1) as a template, the reaction system was prepared according to the standard procedure of the optimized OTRRC method RT-RPA-CRISPR / Cas12a single tube method established in Example 1. The sensitivity of the OTRRC-FD and OTRRC-LFIA fluorescence detection methods and the test strip detection methods using the H1N1 influenza A virus plasmid as a template was analyzed.

[0111] Specifically, prepare Part A—the RT-RPA amplification system (total volume 25 μL): Add 14.75 μL of rehydration buffer, 5.75 μL of DEPC-treated water, 1.00 μL each of forward and reverse primers to achieve a final primer concentration of 480 nM, 1.25 μL of L gene plasmid, half a grain of RPA lyophilized powder, and 1.25 μL of MgOAc to a sterile PCR tube. After adding the components, perform a brief centrifugation (approximately 5–10 s, using a standard microcentrifuge in short centrifugation mode) to collect the liquid at the bottom of the tube, gently mix, and proceed to the next step.

[0112] Configure Part B—CRISPR / Cas12a cleavage system (total volume 10 μL), components and order of addition: 1.6 μL LEPC-treated water, 3.5 μL 100 nM EnGen® Lba Cas12a (Cpf1), 3.5 μL 10×NEBuffer™ r2.1, 0.35 μL 10 μM crRNA (SEQ ID NO.6), 1.05 μL 300 nM ssDNA (SEQ ID NO.7).

[0113] Finally, the RPA amplification was completed by incubation at 37°C for 15 min. The CRIPSR / Cas12a cleavage system on the tube cap was centrifuged to the bottom of the tube and thoroughly mixed with the RPA amplification product. The mixture was then placed on a qPCR instrument and reacted at 37°C for 30 min, with fluorescence signals collected every 30 s.

[0114] The results are as follows Figure 9 As shown; indicating a value as low as 2.8 × 10⁻⁶.-1 Copies / μL of H1N1 influenza A virus can be detected by the optimized OTRRC-FD method.

[0115] After diluting the amplification product with 25 μL DEPC-treated water, take a lateral immunochromatographic test strip coated with colloidal gold-anti-FAM antibody (sample pad), goat anti-mouse secondary antibody (detection line), and avidin (control line), and insert the sample pad end down into the diluted reaction solution (the liquid level should not exceed the Max line). After standing at room temperature for 5 minutes, remove the test strip and read the results flat.

[0116] The results are as follows Figure 10 As shown (Note: The template concentration of 2.8 × 10⁻¹ copies / μL is not shown in Figure 10 because it is below the visual detection limit of the lateral immunochromatographic assay (LFIA) strip, and its signal intensity is insufficient to form a recognizable band on the strip). The results demonstrate that the template concentration is as low as 2.8 × 10⁻¹ copies / μL. 1 The optimized OTRRC-LFIA method can detect H1N1 influenza A virus at a concentration of copies / μL. This demonstrates that H1N1 influenza A virus can be detected even when the laboratory lacks a quantitative fluorescence amplification instrument or when only a water bath is available outdoors.

[0117] 2. Sensitivity detection using fake viruses as templates

[0118] 200 μL each of QuickExtract DNA Extraction Solution and H1N1 influenza pseudovirus were mixed in equal volumes, centrifuged, and then incubated in a dry incubator at 95°C for 5 min to release viral RNA, yielding 1.71 × 10⁻⁶ PCR results. -1 1.71×10 0 1.71×10 1 copies / μL (3 concentrations) of H1N1 influenza pseudovirus RNA.

[0119] Specifically, prepare Part A—RT-RPA amplification system (total volume 25 μL). Add the following components sequentially to a sterile PCR tube: 14.75 μL rehydration buffer, 5.75 μL DEPC-treated water, 1.00 μL each of forward and reverse primers (both at 480 nM), 1.25 μL pseudoviral RNA, half a grain of RPA lyophilized powder, 1.25 μL MgOAc, 0.24 μL 200 U / μL SuperScript™ IV reverse transcriptase, 0.48 μL 5 U / μL LRNase H, and 0.5 μL LTT. After brief centrifugation to mix, proceed immediately to the next step.

[0120] Configure Part B—CRISPR / Cas12a cleavage system (total volume 10 μL), components and order of addition: 1.6 μL LEPC-treated water, 3.5 μL 100 nM EnGen® Lba Cas12a (Cpf1), 3.5 μL 10×NEBuffer™ r2.1, 0.35 μL 10 μM crRNA, 1.05 μL 300 nM ssDNA.

[0121] Finally, the RPA amplification was completed by incubation at 37°C for 15 min. The CRIPSR / Cas12a cleavage system on the tube cap was centrifuged to the bottom of the tube and thoroughly mixed with the RPA amplification product. The mixture was then placed on a qPCR instrument and reacted at 37°C for 30 min, with fluorescence signals collected every 30 s.

[0122] It should be noted that when analyzing the sensitivity of the OTRRC-FD and OTRRC-LFIA fluorescence detection methods and test strip detection methods using H1N1 influenza virus pseudovirus as a template, different concentrations of pseudovirus RNA were used as templates to replace the plasmid in the original system. The RNA reaction system was basically the same as the plasmid detection system described above, except that 0.24 μL of 200 U / μL SuperScript™ IV reverse transcriptase, 0.48 μL of 5 U / μL RNase H, and 0.5 μL of LTT were added to component A, and pseudovirus RNA was used instead of the plasmid. After the reaction, the fluorescence signal was collected using a qPCR instrument, or a portion of the reaction product was taken for detection using a chromatography test strip.

[0123] The collected fluorescence intensity results are as follows Figure 11 As shown, the sensitivity of the OTRRC method, analyzed using pseudoviruses as templates, was as low as 1.7 × 10⁻⁶. 0 Influenza A virus RNA at a concentration of copies / μL can be detected by fluorescence assay.

[0124] The sensitivity of the OTRRC-LFIA method is as low as 10. 1 The pseudovirus of H1N1 influenza A at a concentration of copies / μL can be detected by chromatographic test strips, indicating that it can be visually detected by the naked eye. 1 The H1N1 influenza pseudovirus (copies / μL) can be detected within 35 minutes using only a simple constant-temperature water bath without the need for special instruments.

[0125] Example 3

[0126] This embodiment verifies the specificity of the human H1N1 influenza A detection kit provided in Example 1. The method is as follows:

[0127] Pseudovirus templates (10) were used to target H1N1 influenza A and four other pathogens causing respiratory infections with the same or similar symptoms (FluA-H1N1, SARS-CoV-2, FluB, RSV-A). 2 The results were measured in copies / μL, with DEPC-treated water used instead of pseudovirus in the negative group. The reaction system was prepared according to the standard procedure of the OTRRC method RT-RPA-CRISPR / Cas12a single-tube method established in Example 1. The specificity of the OTRRC-FD and OTRRC-LFIA fluorescence detection methods and the test strip detection methods was analyzed.

[0128] Specifically, the A-part RT-RPA amplification system (total volume 25 μL) was prepared by adding the following components sequentially to a sterile PCR tube: 14.75 μL rehydration buffer, 5.75 μL DEPC-treated water, 1.00 μL each of forward and reverse primers (final concentration 480 nM), 1.25 μL viral template, half a grain of RPA lyophilized powder, and 1.25 μL MgOAc; 0.24 μL 200 U / μL SuperScript™ IV reverse transcriptase, 0.48 μL 5 U / μL RNase H, and 0.5 μL LTT. After brief centrifugation and mixing, proceed to the next step immediately. NTC served as a negative control, replacing the pseudovirus template with sterile water.

[0129] Configure Part B—the CRISPR / Cas12a digestion system (total volume 10 μL), with the following components and order of addition: 1.6 μL LEPC-treated water, 3.5 μL 100 nM EnGen® Lba Cas12a (Cpf1), 3.5 μL 10×NEBuffer™ r2.1, 0.35 μL 10 μM crRNA, and 1.05 μL 300 nM ssDNA. The RNA reaction system is basically the same as the plasmid detection system described above, except that pseudoviral RNA is added to Part A instead of the plasmid.

[0130] Finally, RPA amplification was completed by incubation at 37℃ for 15 min. The CRIPSR / Cas12a cleavage system on the tube cap was centrifuged to the bottom of the tube and thoroughly mixed with the RPA amplification product. The mixture was then incubated at 37℃ for 30 min using a qPCR instrument, with fluorescence signals collected every 30 s. Simultaneously, a portion of the reaction product was tested using lateral immunochromatographic assay strips. The results showed that the constructed method only detected the target pathogen (strong fluorescence signal at the endpoint of the H1N1 influenza A virus reaction). There was no statistically significant difference in fluorescence signal values ​​between the other three viruses and the negative group. Figure 12 As shown.

[0131] The OTRRC-LFIA method for H1N1 influenza A virus established in this invention exhibits good specificity and shows no cross-reactivity with other non-target respiratory tract infection viruses. Results are as follows... Figure 13 As shown.

[0132] Example 4

[0133] This embodiment uses the human H1N1 influenza A detection kit provided in Example 1 for sample verification and detection. The detection method is as follows:

[0134] Twenty H1N1 influenza A virus samples, five novel coronavirus samples, five influenza B virus Victoria lineage samples, and five respiratory syncytial virus RNA samples were analyzed using the OTRRC-FD method, the OTRRC-LFIA method, the RT-RPA-CRISPR / Cas12a single-tube fluorescence method, and the lateral immunochromatographic strip method. The consistency with the results of the nucleic acid detection "gold standard" RT-qPCR was evaluated.

[0135] The specific operation is the same as in Example 3, except that the viral template is replaced with an RNA sample.

[0136] Sample performance evaluation results are as follows Figure 14 As shown, the sensitivities of the OTRRC-FD method and the OTRRC-LFIA method for detecting H1N1 influenza virus in real samples were 95% and 90%, respectively; the specificity of both methods was 100%; and the consistency with RT-qPCR was 97.19% and 94.56%, respectively.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A nucleic acid genome for detecting influenza A (H1N1) virus, characterized in that, It includes an RPA amplification primer pair, crRNA, and an ssDNA probe, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of the crRNA is shown in SEQ ID NO.6, and the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO.

7.

2. The nucleic acid genome as described in claim 1, characterized in that, The nucleic acid group also includes a positive control template nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO.

1. This positive control template nucleic acid molecule contains a binding site for the RPA amplification primer pair and a recognition site for crRNA.

3. A kit for detecting influenza A (H1N1) virus, characterized in that, It includes the nucleic acid genome as described in claim 1 or 2.

4. The kit as described in claim 3, characterized in that, It also includes lateral immunochromatographic test strips, wherein the sample pad of the lateral immunochromatographic test strip is coated with colloidal gold-labeled anti-FAM monoclonal antibody, the detection line is coated with goat anti-mouse secondary antibody, and the control line is coated with avidin. The lateral immunochromatographic test strip determines whether the Cas12a enzyme is activated by the presence or absence of the test line, and determines the validity of the test strip by the presence or absence of the control line. The specific criteria are as follows: if both the control line and the test line are colored, the sample is positive; if the control line is colored but the test line is not colored, the sample is negative; if the control line is not colored, the test strip is invalid and the test result is invalid.

5. The kit according to claim 4, characterized in that, The two ends of the ssDNA probe are labeled with the fluorescent groups FAM and Biotin, respectively.

6. The kit as described in claim 4 or 5, characterized in that, The kit also includes RPA lyophilized powder, rehydration buffer, DEPC-treated water, MgOAc, reverse transcriptase, RNaseH, DTT, and Cas12a protein; the RPA lyophilized powder contains recombinase, single-stranded binding protein SSB, and DNA polymerase.

7. The use of the nucleic acid genome according to claim 1 or 2 in the preparation of a visual detection reagent for detecting influenza A (H1N1) virus.

8. A visual reagent for detecting influenza A (H1N1) virus, characterized in that, It includes detection reagents and display reagents. The detection reagents include a bottom reagent placed at the bottom of the reaction tube and a cap reagent placed inside the cap of the same reaction tube. The bottom reagent is an RPA amplification system containing primer pairs with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.

4. The cap reagent is a CRISPR / Cas12a cleavage system containing crRNA with nucleotide sequence as shown in SEQ ID NO.6, an ssDNA probe with nucleotide sequence as shown in SEQ ID NO.7, Cas12a protein, and buffer. The ssDNA probe is modified with fluorescent groups X and Biotin at both ends, respectively. The display reagent is a lateral immunochromatographic test strip. The sample pad of the lateral immunochromatographic test strip is coated with a colloidal gold-labeled monoclonal antibody against fluorescent group X, the detection line is coated with goat anti-mouse secondary antibody, and the control line is coated with avidin. The lateral immunochromatographic test strip determines whether the Cas12a enzyme is activated by the presence or absence of color development on the detection line. The fluorescent group X is any one of FAM, HEX, Red, Cy3, Cy5, Cy7, FITC, JOE, TET, and VIC.

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