Influenza virus H1N1 lateral flow analysis biosensor and application thereof

Abstract

The invention provides an influenza virus H1N1 lateral flow analysis biosensor and application thereof, and belongs to the field of detection. The invention provides an influenza virus H1N1 lateral flow analysis biosensor. The influenza virus H1N1 lateral flow analysis biosensor comprises a primer Primer-9, a template Template-9, a Bst DNA (Deoxyribose Nucleic Acid) polymerase, dNTPs (Deoxyribose Nucleic Acid), a PBS (Phosphate Buffer Solution), an FAM Probe and an LFA test strip. The biosensor provided by the invention can be applied to preparation of a product for detecting and judging the influenza virus H1N1, and compared with a traditional detection method, the biosensor provided by the invention can obtain relatively high amplification efficiency by only needing one primer and template, is simpler, more convenient and quicker to operate, and relatively high in accuracy and sensitivity, and can be applied to detection of the influenza virus H1N1. The problems that in the prior art, the H1N1 virus detection process is complex, and in the prior art, the isothermal technology is limited are solved.

Description

Technical Field

[0001] The invention belongs to the field of detection, and in particular relates to a biosensor for detecting influenza virus H1N1. Background Art

[0002] Nucleic acids store genetic information that encodes proteins and are also important biomarkers for early disease diagnosis and food safety testing. However, nucleic acids are often not directly detectable due to their low concentration. Therefore, amplification methods are essential to increase the nucleic acid content to detectable levels. Polymerase chain reaction (PCR) is widely considered the gold standard for amplifying and detecting nucleic acids of pathogenic microorganisms. However, PCR requires expensive thermal cycling instruments and trained professionals. To overcome this limitation, several isothermal amplification techniques have been developed as alternatives to PCR that require constant temperature rather than repeated thermal cycling, thereby eliminating the need for expensive and bulky thermal cycling instruments and avoiding the complex amplification steps required for PCR, such as high-temperature denaturation, annealing, and extension. Over the past two decades, isothermal methods for nucleic acid amplification have been developed as PCR alternatives for detecting pathogenic microorganisms, such as recombinant polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification. Ganguli et al. developed a reverse transcription-LAMP isothermal detection method for the detection of severe acute respiratory syndrome coronavirus; Lim et al. constructed a reverse transcription-LAMP-based point-of-care (POC) device for the simultaneous detection of four respiratory viruses (SARS CoV-2, influenza A, influenza B, and respiratory syncytial virus); Ahamed et al. used RPA technology to establish a CRISPR-Cas12a-assisted nanopore for the detection of monkeypox virus. However, the existing RPA technology requires multiple enzymes for isothermal amplification of nucleic acids, such as auxiliary proteins, single-stranded (ss) binding proteins, and strand displacement polymerases, resulting in a cumbersome detection process. Therefore, the application of existing isothermal amplification technology in clinical diagnosis is limited. Therefore, it is necessary to develop a new isothermal amplification method to solve the above dilemma.

[0003] Due to the antigenic differences of hemagglutinin and neuraminidase on the influenza A virus envelope, influenza A virus can be further divided into different subtypes. Therefore, establishing a suitable detection method is crucial for the early diagnosis and early detection of H1N1 influenza virus. In the prior art, PCR and other methods are often used for detection, and the detection process is relatively complicated, so an accurate and convenient method for detecting influenza virus H1N1 is urgently needed. Summary of the invention

[0004] The present invention provides an influenza virus H1N1 lateral flow analysis biosensor (hereinafter referred to as SPA-LFA) which solves the limitations of the isothermal amplification technology in the prior art and also solves the problem of the relatively complicated process of detecting H1N1 virus in the prior art.

[0005] A lateral flow analysis biosensor for influenza virus H1N1, wherein the biosensor uses a SPA isothermal amplification method for detection, and the biosensor comprises: a primer Primer-9, a template Template-9, a Bst DNA polymerase, dNTPs, a PBS buffer, a FAM Probe and an LFA test strip.

[0006] Preferably, the sequence of Primer-9 is TCTCTTATT.

[0007] Preferably, the sequence of Template-9 is AATAAGAGAAATAAGAGA-3InvdT.

[0008] Preferably, the sequence of the FAM Probe is 5FAM-GACAAGACCAATCCT.

[0009] Preferably, the Bst DNA polymerase is 4 U.

[0010] Preferably, the dNTPs are prepared by mixing dTTP and UTP-Biotin at a final concentration ratio of 1:1.

[0011] Preferably, the pH of the PBS buffer is 7.

[0012] The above biosensor is used in the preparation of a product for detecting and judging influenza virus H1N1, and the application method comprises the following steps: S1: The reaction system is 35 μL, which includes 2 μL 5 μM dNTPs, 10 μL 600 nM Primer-9, 10 μL 300 nM Template-9, 2 μL 4 U Bst DNA polymerase, 5 μL PBS buffer and 6 μL enzyme-free water. After mixing evenly, react at 55 °C for 1-4 h, and then inactivate the enzyme at 80 °C to obtain SPA product; S2: 2 μL FAM-Probe, 5 μL SPA product and 28 μL PBS buffer were mixed into a 35 μL reaction system, the sample to be tested was added into the reaction system, and the mixture was obtained by incubation at 37 °C for 10 min; S3: Add the mixture to the LFA test strip and add 25 μL PBS, then observe. If red lines appear at both the Control and Test, the sample to be tested is positive, otherwise it is negative.

[0013] Preferably, the concentration of the FAM-Probe is 1.5 μM.

[0014] Preferably, the detection limit of the sample to be detected is 18 pM.

[0015] Beneficial Effects The present invention establishes a method for detecting H1N1 virus by SPA-LFA. The extension amplification of primers is achieved by the special design of nucleotide sequences. Compared with the common isothermal amplification technology which requires multiple primers and multiple enzymes, the detection method of the present invention only requires one primer and template to obtain a higher amplification efficiency. In addition, this study combines the established SPA isothermal amplification method with the LFA test strip. The quality control line (C) and the detection line (T) of the LFA test strip can be observed as a red strip signal by naked eyes at room temperature. Therefore, the detection method provided by the present invention is more simple and intuitive, and can be portable and rapid for detecting H1N1 virus, and has great potential for POC diagnosis and clinical analysis.

[0016] The present invention provides a lateral flow analysis biosensor for influenza virus H1N1. The SPA product ssDNA can be prepared in advance and directly detected with the sample RNA and related probes to be tested, without the need for complex separation steps or temperature adjustment procedures. The time from collecting samples to obtaining final results is less than 1 hour. It is worth noting that it only takes 10 minutes from incubation to obtaining results using simple equipment, while quantitative PCR takes 1 to 2 hours, so the method provided by the present invention is simpler and faster.

[0017] By using an influenza virus H1N1 lateral flow analysis biosensor provided by the present invention to detect spiked clinical serum and saliva samples, as well as chicken embryo allantoic fluid infected with the H1N1 virus (simulating the H1N1 virus in clinical samples), the results are consistent with the results of the enzyme-linked immunosorbent assay (ELISA), demonstrating that the biosensor has practical application capabilities and high accuracy.

[0018] The influenza virus H1N1 lateral flow analysis biosensor provided by the present invention has high sensitivity. The SPA method established by the present invention has high amplification efficiency and short reaction incubation time, which can effectively reduce the degradation of target RNA, thereby improving the sensitivity of the biosensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagrams, where A is the schematic diagram of the SPA isothermal amplification system; B is the schematic diagram of the SPA-LFA; Figure 2 The SPA characterization results are shown in Figure 1, where A is the atomic force microscope (AFM) image of the SPA product; B is a local magnified image of A; Figure 3 It is the AFM height distribution diagram of SPA products, where A is the AFM image of SPA product height distribution; B is the three-dimensional height profile diagram; Figure 4 It is the PAGE gel image of SPA product, where 9, 11, 13 and 15 are the base composition numbers of primers, and the red and green fluorescent bands represent the Cy3-labeled primer (P) and the Cy5-labeled template (T), respectively. Lane 1: T9 (1mm); Lane 2: P9 (1mm); Lane 3: T9 and P9 (1mm); Lane 4: T11 (1mm); Lane 5: P11 (1mm); Lane 6: T11 and P11 (1mm); Lane 7: T13 (1mm); Lane 8: P13 (1mm); Lane 9: T13 and P13 (1mm); Lane 10: T15 (1mm); Lane 11: P15 (1mm); Lane 12: T15 and P15 (1mm); Figure 5 To verify the detection results of SPA-LFA for H1N1 virus, A is the visual detection result diagram when a single component is missing; B is the detection line to quality control line (T / C) result diagram of SPA-LFA when a single component is missing; Figure 6 The results of SPA-LFA condition optimization are shown in Figure 1, where A is the dTTP / UTP-Biotin ratio optimization result diagram; B is the FAM probe concentration optimization result diagram; C is the incubation time optimization result diagram; and D is the buffer pH optimization result diagram; Figure 7 This is the temperature optimization result diagram for SPA; Figure 8 Figure 1 shows the sensitivity results of SPA-LFA, where A shows the detection results at different concentrations; B shows the peak T / C ratio and the H1N1 concentration from 5 to 5 × 10 5 pM linear relationship equation, where the abscissa is the logarithm of the H1N1 concentration (pM) in the sample to be tested (lg(H1N1)(pM)), and the ordinate is the peak T / C ratio of the test strip; Fig. 9 SPA-LFA specificity result diagram, wherein A is the detection result of different subtypes of influenza virus; B is the T / C ratio of SPA-LFA of different subtypes of influenza virus; Fig.10The results of the cross-reactivity test of SPA-LFA against influenza A (H1N1) virus and other subtypes of influenza virus are shown in Figure 1, where A is the visual test result; B is the T / C value bar graph, where C represents the quality control line; T represents the test line; Fig.11 The actual sample analysis results are shown in Figure 1, where A is the SPA-LFA visualization test result; B is the peak area of ​​the T / C value of each group, I: 1.0 × 10 6 copies / mL H1N1; II:1.0 × 10 5 copies / mL H1N1; III:1.0 ×10 4 copies / mL H1N1. DETAILED DESCRIPTION

[0020] Example 1. Visual lateral flow analysis biosensor and experimental method for influenza virus H1N1.

[0021] 1. Design principle of SPA-LFA.

[0022] The working principle of SPA-LFA is based on the sensing of the linear amplification of the target H1N1. Figure 1 As shown, the proposed SPA-LFA includes two steps: (1) obtaining SPA products, which includes SPA isothermal amplification reaction of two partially complementary DNA fragments. Figure 1 As shown in A, the reaction includes a long template probe Template and a short primer probe Primer-H1N1. Primer-H1N1 consists of two parts. The proximal fragment located in the 3′ end domain is complementary to the template probe Template and is used for initial amplification; the fragment located in the 5′ end domain is used as the target recognition fragment and is complementary to part of the H1N1 sequence. (2) The conjugate SPA for detecting LFA, the principle is as follows Figure 1As shown in B. First, Bio-11-UTP (U-Biotin) and the prepared SPA product ssDNA were added, and in the presence of the target H1N1 RNA, a complex consisting of the FAM probe, the target H1N1 RNA, and the SPA product ssDNA (SPA-ssDNA) was formed. The specific base length of the target H1N1 RNA is about 20 bp, of which 10 bp is complementary to the 3' end of the FAM probe and the other 10 bp is complementary to the 5' end of the SPA-ssDNA. Finally, the formed "FAM-Biotin / target H1N1 / SPA product" triple complex binds to the SA-AuNPs (gold nanoparticles labeled with streptavidin) on the test strip through the high recognition efficiency of streptavidin SA and biotin Biotin. Subsequently, the complex was detected by immunorecognition of the anti-Fam monoclonal antibody embedded in the T line. Then, the excess SA-AuNPs were captured on the quality control line C line to form another red band. Finally, semi-quantitative detection was performed by the color depth of the detection line T line. When there is no target H1N1 RNA, the "FAM-Biotin / target H1N1 / SPA product" complex cannot be formed, and no red band appears on the T line. The entire operation process does not require magnetic separation and washing steps and variable temperature environment, and rapid detection visible to the naked eye can be achieved at room temperature.

[0023] 2. Oligonucleotides used in this study are shown in Table 1.

[0024] Table 1

[0025] 3. Composition of the influenza virus H1N1 visual lateral flow analysis biosensor.

[0026] Primer-9, template-9, Bst DNA polymerase, dNTPs, PBS buffer, FAM Probe, LFA test strip.

[0027] 4. SPA isothermal amplification was used to obtain biotin-labeled ssDNA.

[0028] Biotin-labeled ssDNA is generated by adding biotin-modified uridine triphosphate. The 35 µL reaction system includes: 2 μL of dNTPs (5 μM; the ratio of dTTP to UTP-Biotin is 1:1), 10 μL of Primer-9 (600 nM), 10 μL of Template-9 (300 nM; 3' end modified with InvdT), 2 μL of Bst DNA polymerase (4 U), 5 μL of PBS buffer and 6 μL of enzyme-free water. After thorough mixing, react at 55 °C for 1-4 h, and inactivate the enzyme at 80 °C. The biotin-labeled SPA product ssDNA is obtained. In order to increase the detection speed, ssDNA can be prepared in advance and stored at 4 °C for later use.

[0029] 5. H1N1 detection method.

[0030] The influenza A virus (A / Puerto Rico / 8-SV14 / 1934(H1N1)) gene sequence (CY147535.1) was downloaded from the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ), and H1N1 RNA was synthesized by Sangon Biotech. 2 μL FAM-Probe (1.5 μM), 5 μL SPA product ssDNA and 28 μL PBS buffer were mixed into a 35 μL reaction system, and the sample to be tested (H1N1 RNA) was added to the reaction system, and incubated at 37°C for 10 min to form a FAM-Biotin biotin mixture. The above mixture was added dropwise to the LFA test strip, and 25 μL PBS was added continuously. The FAM-Biotin / target H1N1 / SPA product in the mixture was bound to the colloidal gold nanoparticles and streptavidin (SA-AuNPs) on the binding pad on the LFA test strip and assembled under capillary action. Then, the FAM-Biotin / target H1N1 / SPA product was captured by the anti-Fam antibody fixed on the T detection line, resulting in the generation of red bands. Preliminary evaluation was performed by naked eye. About 3 minutes after adding the sample, images were taken with a smartphone and analyzed and quantified with ImageJ software.

[0031] 6. Statistical analysis.

[0032] ImageJ software was used to quantify the signal intensity of the nucleic acid test paper. The signal intensity was expressed as the T / C ratio, where T was the peak area of ​​the detection line signal and C was the peak area of ​​the quality control line signal. Three sets of parallel measurements were performed for each sample. The T / C value in the figure is expressed as the mean ± standard deviation (SD) of three sets of parallel measurements. Prism 8 software was used for statistical analysis.

[0033] Example 2. Primer screening and feasibility analysis.

[0034] 1. Primer screening.

[0035] In order to determine the most efficient primers in the biosensor (SPA-LFA), four pairs of primers and templates were set: Primer-9 / Template-9, Primer-11 / Template-11, Primer-13 / Template-13, and Primer-15 / Template-15. The numbers 9, 11, 13, and 15 represent the base numbers of the primers. The above primers were used for SPA amplification to obtain SPA products. The SPA products were characterized by atomic force microscopy. The morphology of the SPA products was a typical DNA chain of 3.51±0.04 nm (e.g. Figure 2 A and B), and Figure 4 The PAGE results in the table above correspond to those in the table above. Figure 3 From the three-dimensional height profiles of SPA products in A and B, it can be seen that SPA products have a certain theoretical length. Although DNA may break into short fragments different from the theoretical length, most DNA sequences conform to the expected length, which indicates that the SPA extension process can change the DNA sequence from the nanometer scale to the micrometer scale.

[0036] The feasibility of SPA isothermal amplification reaction was confirmed by polyacrylamide gel electrophoresis (PAGE) of SPA products and imaging and analysis using ImageJ software, and the amplification effect of SPA was further demonstrated. First, four pairs of primers and templates (5′ end) were labeled with fluorescent markers Cy5 and Cy3 respectively to verify the efficiency of SPA. Figure 4 As shown in the figure, regardless of the primer length, SPA can successfully amplify when the primer and template are present at the same time, indicating that the above 4 pairs of primers and templates are universal. However, when Primer-9 / Template-9 is used, the amplification efficiency of the signal in lane 3 is relatively strong. Therefore, we chose Primer-9 / Template-9 as the reaction primer and template for subsequent experiments. Overall, the results prove the effectiveness of the SPA system.

[0037] 2. Feasibility analysis

[0038] The feasibility of the influenza virus H1N1 visualized lateral flow analysis biosensor was verified by designing a deletion experiment on the components of the influenza virus H1N1 visualized lateral flow analysis biosensor. Figure 5 As shown in A and B, the peak T / C ratio will increase significantly only when all components are present; the absence of one component will result in no positive signal on the T line.

[0039] Example 3. Optimization of reaction conditions.

[0040] The amplification efficiency of the SPA isothermal amplification reaction is a key factor affecting the performance of the established biosensor. In other words, the more ssDNA produced by the SPA product, the more binding sites it has, which can be more efficiently connected to the FAM-Probe probe, thereby improving the detection ability of the established biosensor.

[0041] Optimize the ratio of dTTP to UTP-Biorin. Figure 6 As shown in A, the optimal ratio of unlabeled dTTP to UTP-Biotin is 1:1. This may be because too low a concentration of UTP-Biotin will cause the SPA product ssDNA to have too few Bition sites, thus affecting the final binding efficiency with the test strip, while too high a concentration of UTP-Biotin will produce steric hindrance, reducing the amplification efficiency of SPA and reducing the amount of SPA product ssDNA.

[0042] Optimize the concentration of FAM-Probe probe. Optimize the concentration of FAM-Probe probe under the condition of the best ratio of dTTP and UTP-Biotin. The SPA product ssDNA can provide binding sites for the FAM-Probe probe and the target H1N1 virus RNA. The three can form a triple nucleic acid complex (FAM-Biotin / target H1N1 / SPA product), triggering the signal generation on the T line of the lateral flow analysis test strip. However, excessive FAM-Probe probe may affect the binding efficiency of the complex (FAM-Biotin / target H1N1 / SPA product) on the test strip. At the same time, excessive free FAM-Probe probe will also cause unnecessary waste of reagents and increase costs. Therefore, an appropriate concentration of FAM-Probe probe can improve the detection performance of SPA-LFA. Figure 6 As shown in Figure B, as the concentration of the FAM-Probe probe increases, the peak T / C ratio increases and reaches the optimal ratio at 1.5 µM. Therefore, 1.5 µM was selected as the optimal concentration of the FAM-Probe probe.

[0043] Optimize the incubation time for H1N1 detection. Optimize the incubation time under the above optimal conditions. Figure 6 As shown in Figure 3, the performance of SPA-LFA did not change significantly during the incubation time of 5 to 60 min. Therefore, 10 min was selected as the optimal incubation time in subsequent studies. The total time required for SPA-LFA detection was about 40 min, including 30 min for RNA extraction and 10 min for detection.

[0044] Optimize the pH value of PBS buffer for H1N1 detection. Under the above optimal conditions, optimize the pH value of the buffer. Figure 6 As shown in D, the performance of SPA-LFA is relatively stable in the pH range of 5.4 to 9.4. From the perspective of biological environmental adaptability, PBS buffer with a pH of 7 is the best choice.

[0045] Optimize SPA reaction temperature. Under the above optimal conditions, the SPA reaction temperature was optimized to further improve the efficiency of SPA. The SPA reaction was carried out in the temperature range of 45~65 ℃. Figure 7 As shown in the figure, the peak T / C results show that the strongest signal appears at 55 ℃, so the optimal reaction temperature is 55 ℃.

[0046] Example 4. Sensitivity analysis of the biosensor (SPA-LFA).

[0047] In order to evaluate the sensitivity of the proposed biosensor, under the optimal experimental conditions of Example 3, the samples to be tested were 5 ~ 5×10 5 The samples were diluted 10 times in the pM range and then evaluated. ImageJ software was used to analyze the relationship between the peak T / C ratio of the test strip and the different concentrations of the samples. The results are shown in Figure 2. Figure 8 The color change and signal intensity of the T line of the test strip increase with the increase of the concentration of the target nucleic acid fragment of the influenza A (H1N1) virus in the sample to be tested ( Figure 8 The peak T / C ratio increases with the H1N1 concentration from 5 to 5 × 10 5 pM grows logarithmically ( Figure 8 The linear relationship between the concentration of the sample to be tested and the test result is Y = 0.6171X - 0.8079 (R 2 = 0.9514), where Y is the peak T / C ratio of the test strip and X is the logarithm of the H1N1 concentration (pM) in the sample to be tested. The calculated limit of detection (LOD) is 18 pM (3α / S, where α is the standard deviation of the blank solution and S is the slope). Figure 8 As shown in A, the LOD of naked eye detection is 50 pM. In addition, the biosensor of the present invention shows a higher advantage in detecting HIN1 virus compared with other biosensors, as shown in Table 2.

[0048] Table 2

[0049] Example 5. Specific detection of biosensors.

[0050] To verify the specificity of the biosensor for detecting HIN1 virus, enzyme-free water was used as a negative control to detect RNA of different subtypes of influenza viruses (H3N2, H5N1, H6N2, H7N9 and H9N2). The sample concentration used was 1 × 10 5 pM was used to evaluate the specificity of SPA-LFA. Fig. 9 As shown in the figure, SPA-LFA was positive only for HIN1 virus, but negative for the other five viruses, indicating that SPA-LFA has good specificity for HIN1 virus. In addition, the potential cross-reactivity of SPA-LFA for other influenza virus subtypes (H3N2, H5N1, H6N2, H7N9 and H9N2) was also studied. Fig.10 As shown, there was little cross-reactivity between the target H1N1 virus and other subtypes, further confirming the excellent selectivity of SPA-LFA for HIN1 virus.

[0051] Example 6. Spiked recovery experiment.

[0052] The spike recovery experiments were performed using serum and saliva samples to evaluate the ability of the biosensor of the present invention to detect H1N1 in real complex biological samples and to determine whether the biosensor is affected by external interference factors. First, 10% serum was treated with 0.2U / μL of RNase inhibitor (to eliminate the effect on the target RNA). Subsequently, the samples were centrifuged at 6000 rpm for 20 minutes and then filtered through a 30K ultrafiltration centrifuge tube to remove biomolecules. The H1N1 RNA was diluted to 10% with the treated 10% serum. 3 , 10 4 and 10 5 pM for recovery analysis. In this experiment, 10% biological serum without H1N1 RNA was used as a negative control. At the same time, the saliva samples of healthy individuals were spiked with the same concentration of H1N1 RNA (10 3 , 10 4 and 10 5 pM) was used to evaluate the recovery rate, and saliva samples without H1N1 RNA were used as negative controls.

[0053] Then, the biosensor (SPA-LFA) of the present invention was used for detection and analysis, and the target H1N1 was detected, verifying the applicability of the method. As shown in Table 3, the SPA-LFA recovery rate of saliva and serum samples was 93.8% to 130.7%, and the acceptable relative standard deviation was less than 11.1%. In general, these results confirm the applicability of the biosensor (SPA-LFA) of the present invention for H1N1 virus detection in field and clinical settings.

[0054] Table 3

[0055] Example 7. Verification of actual sample detection effect.

[0056] In order to evaluate the feasibility of the biosensor (SPA-LFA) of the present invention on actual samples, viral RNA was isolated from the allantoic fluid of chicken embryos infected with H1N1 using a viral RNA extraction kit. The total viral RNA was quantified using an ultraviolet spectrophotometer and stored at -80°C. The detection concentration was 1.0 × 10 4 ~ 1.0 × 10 6 copies / mL, visualized by naked eye or grayscale analysis using ImageJ software, the results are as follows Fig.11 As shown, the practicality of the proposed biosensor (SPA-LFA) was explored. Compared with commercial influenza virus test strips, commercial test strips usually detect antigens as targets, which require long-term infection and high concentrations to determine positive results. This method can directly detect H1N1 virus RNA. In the detection of different concentrations of RNA, H1N1 virus was accurately detected in all samples, and the signal intensity was obvious. In contrast, no signal was detected on the detection T line of the uninfected chicken embryo allantoic fluid sample.

[0057] Eight samples were tested simultaneously using the commercial gold standard ELISA kit and biosensor (SPA-LFA), of which samples 1 and 2 were the same negative and non-infected chicken embryo allantoic fluid at different periods, and samples 3-8 were chicken embryo allantoic fluid infected with different H1N1 concentrations. The results are shown in Table 4, indicating that the two methods showed consistency, further proving the application capability of the biosensor (SPA-LFA) detection method in actual sample detection.

[0058] Table 4

Claims

1. A lateral flow analysis biosensor for influenza virus H1N1, characterized in that: The biosensor uses the SPA isothermal amplification method for detection, and the biosensor comprises: primer Primer-9, template Template-9, Bst DNA polymerase, dNTPs, PBS buffer, FAM Probe and LFA test strip.

2. The biosensor according to claim 1, characterized in that: The sequence of Primer-9 is TCTCTTATT.

3. The biosensor according to claim 1, characterized in that: The sequence of Template-9 is AATAAGAGAAATAAGAGA-3InvdT.

4. The biosensor according to claim 1, characterized in that: The sequence of the FAM Probe is 5FAM-GACAAGACCAATCCT.

5. The biosensor according to claim 1, characterized in that: The amount of the Bst DNA polymerase was 4 U.

6. The biosensor according to claim 1, characterized in that: The dNTPs are prepared by mixing dTTP and UTP-Biotin at a final concentration ratio of 1:

1.

7. The biosensor according to claim 1, characterized in that: The pH of the PBS buffer is 7.

8. Use of the biosensor according to any one of claims 1 to 7 in the preparation of a product for detecting and judging influenza virus H1N1, characterized in that: The application method comprises the following steps: S1: The reaction system is 35 μL, which includes 2 μL 5 μM dNTPs, 10 μL 600 nM Primer-9, 10 μL 300 nM Template-9, 2 μL 4 U Bst DNA polymerase, 5 μL PBS buffer and 6 μL enzyme-free water. After mixing evenly, react at 55 °C for 1-4 h, and then inactivate the enzyme at 80 °C to obtain SPA product; S2: 2 μL FAM-Probe, 5 μL SPA product and 28 μL PBS buffer were mixed into a 35 μL reaction system, the sample to be tested was added into the reaction system, and the mixture was obtained by incubation at 37 °C for 10 min; S3: Add the mixture to the LFA test strip and add 25 μL PBS, then observe. If red lines appear at both the Control and Test, the sample to be tested is positive, otherwise it is negative.

9. The use according to claim 8, characterized in that: The concentration of the FAM-Probe was 1.5 μM.

10. The use according to claim 8, characterized in that: The detection limit of the sample to be detected is 18 pM.

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