An influenza A (H1N1) lateral flow assay biosensor and its application

Abstract

The present invention provides an influenza A (H1N1) virus lateral flow assay biosensor and its application, belonging to the field of detection. The influenza A (H1N1) virus lateral flow assay biosensor provided by the present invention comprises: primer Primer-9, template Template-9, Bst DNA polymerase, dNTPs, PBS buffer, FAM Probe and LFA test strip. The biosensor provided by the present invention can be used for preparing a product for detecting and judging influenza A (H1N1) virus. Compared with the traditional detection method, it only needs one primer and one template to obtain a higher amplification efficiency, is more convenient and rapid to operate, and has higher accuracy and sensitivity, solving the problems of the relatively complex detection process of H1N1 virus in the prior art and the limitations of isothermal technology in the prior art.

Description

Technical Field

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

[0002] Nucleic acids store genetic information encoding proteins and are also important biomarkers for early disease diagnosis and food safety detection. However, due to the low concentration of nucleic acids, they often cannot be directly detected. Therefore, amplification methods are crucial for increasing the nucleic acid content to a detectable level. Polymerase chain reaction (PCR) is widely regarded as 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, which require a constant temperature instead of repeated thermal cycling, thus eliminating the need for expensive and bulky thermal cycling instruments and avoiding the complex amplification steps required by PCR, such as high-temperature denaturation, annealing, and extension. In the past two decades, isothermal methods of nucleic acid amplification have evolved into alternatives to PCR for detecting pathogenic microorganisms, such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification. Ganguli et al. developed a reverse transcription LAMP isothermal detection method for detecting severe acute respiratory syndrome coronavirus; Lim et al. constructed a point-of-care (POC) device based on reverse transcription LAMP for simultaneous detection of four respiratory viruses (SARS CoV-2, influenza A, influenza B, and respiratory syncytial virus); Ahamed et al. established a CRISPR-Cas12a-assisted nanopore using RPA technology for the detection of monkeypox virus. However, existing RPA techniques require multiple enzymes for isothermal amplification of nucleic acids, such as accessory proteins, single-stranded (ss) binding proteins, and strand displacement polymerases, resulting in a cumbersome detection process. Therefore, the application of existing isothermal amplification techniques in clinical diagnosis is limited. Therefore, it is necessary to develop a new isothermal amplification method to solve the above dilemmas.

[0003] Due to the antigenic differences in hemagglutinin and neuraminidase on the envelope of influenza A virus, influenza A virus can be further divided into different subtypes. Therefore, establishing a suitable detection method is crucial for the early diagnosis and detection of H1N1 influenza virus. In the prior art, methods such as PCR are mostly used for detection, and the detection process is relatively complex. Therefore, there is an urgent need for an accurate and convenient method for detecting influenza virus H1N1. Summary of the Invention

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

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

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

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

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

[0009] Preferably, the amount of 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 application of the above biosensor in the preparation of a product for detecting and judging influenza virus H1N1, the application method includes the following steps:

[0013] S1: The reaction system is 35 μL, which includes 2 μL of 5 μM dNTPs, 10 μL of 600 nM Primer-9, 10 μL of 300 nM Template-9, 2 μL of 4 U Bst DNA polymerase, 5 μL of PBS buffer and 6 μL of enzyme-free water. After mixing evenly, react at 55 °C for 1-4 h, and then inactivate the enzyme at 80 °C to obtain the SPA product;

[0014] S2: Mix 2 μL of FAM-Probe, 5 μL of SPA product and 28 μL of PBS buffer to form a 35 μL reaction system. Add the sample to be detected to the reaction system and incubate at 37 °C for 10 min to obtain a mixture;

[0015] S3: Drop the mixture onto the LFA test strip and add 25 μL of PBS, then observe. If red lines appear at both the Control and Test positions, the sample to be detected is positive; otherwise, it is negative.

[0016] Preferably, the concentration of the FAM-Probe is 1.5 μM.

[0017] Preferably, the detection limit of the sample to be detected is 18 pM.

[0018] Beneficial effects

[0019] The present invention establishes a method for detecting H1N1 virus by SPA-LFA. The extension amplification of the primer is achieved by the special design of the nucleotide sequence. Compared with ordinary isothermal amplification techniques that require multiple primers and various enzymes, the detection method of the present invention only needs one primer and template to obtain a high amplification efficiency. And in this study, the established SPA isothermal amplification method is combined with the LFA test strip. The quality control line (C) and the test line (T) of the LFA test strip can observe red strip signals with the naked eye at room temperature. Therefore, the detection method provided by the present invention is more simple and intuitive, and can detect H1N1 virus in a portable and rapid manner, having great potential for POC diagnosis and clinical analysis.

[0020] For the lateral flow analysis biosensor for influenza virus H1N1 provided by the present invention, the SPA product ssDNA can be pre-prepared and directly detected with the sample RNA to be detected and related probes, without complex separation steps or temperature adjustment procedures. The time from sample collection to obtaining the final result is less than 1 hour. It is worth noting that it only takes 10 minutes from incubation to obtaining the result using a simple device, while quantitative PCR takes 1 - 2 hours. Therefore, the method provided by the present invention is more simple and rapid.

[0021] By using the lateral flow analysis biosensor for influenza virus H1N1 provided by the present invention to detect spiked clinical serum and saliva samples, as well as to detect the allantoic fluid of chicken embryos infected with H1N1 virus (simulating H1N1 virus in clinical samples), the results are all consistent with those of enzyme-linked immunosorbent assay (ELISA), proving that the biosensor has practical application ability and high accuracy.

[0022] The lateral flow analysis biosensor for influenza virus H1N1 provided by the present invention has high sensitivity. The established SPA method has a high amplification efficiency and a short incubation time for the reaction, which can effectively reduce the degradation of target RNA, thereby improving the sensitivity of the biosensor. Brief description of the drawings

[0023] Figure 1Schematic diagrams, where A is the schematic diagram of the SPA isothermal amplification system; B is the schematic diagram of SPA-LFA;

[0024] Figure 2 Diagram of SPA characterization results, where A is the atomic force microscope (AFM) image of the SPA product; B is the partial enlarged view of A;

[0025] Figure 3 Diagram of the atomic force microscope height distribution of the SPA product, where A is the AFM image of the atomic force microscope height distribution of the SPA product; B is the three-dimensional height contour map;

[0026] Figure 4 PAGE gel diagram of the SPA product, where 9, 11, 13, and 15 are the base composition quantities of the 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);

[0027] Figure 5 Diagram of the verification results of SPA-LFA for the detection of H1N1 virus, where A is the visualization 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;

[0028] Figure 6 Diagram of the results of SPA-LFA condition optimization, where A is the optimization result diagram of the dTTP / UTP-Biotin ratio; B is the optimization result diagram of the FAM probe concentration; C is the optimization result diagram of the incubation time; D is the optimization result diagram of the buffer pH value;

[0029] Figure 7 Diagram of the temperature optimization results of SPA;

[0030] Figure 8 Diagram of the sensitivity results of SPA-LFA, where A is the detection results at different concentrations; B is the peak T / C ratio and the linear relationship equation of the H1N1 concentration from 5 to 5 × 10 5 pM. The abscissa is the logarithm (lg(H1N1)(pM)) of the H1N1 concentration (pM) in the test sample, and the ordinate is the peak T / C ratio of the test strip;

[0031] Figure 9 It is the specific result diagram of SPA-LFA, where A is the detection results of different subtypes of influenza viruses; B is the T / C ratio of SPA-LFA for different subtypes of influenza viruses.

[0032] Figure 10 It is the result diagram of the cross-reactivity test of SPA-LFA for influenza A H1N1 virus and other subtypes of influenza viruses, where A is the visual detection result; B is the column chart of T / C values, where C represents the quality control line; T represents the test line.

[0033] Figure 11 It is the result diagram of the actual sample analysis, where A is the visual detection result of SPA-LFA; B is the peak area of T / C values for 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. Specific implementation mode

[0034] Example 1. Visual lateral flow analysis biosensor for influenza virus H1N1 and experimental method.

[0035] I. Design principle of SPA-LFA.

[0036] The working principle of SPA-LFA is based on the sensing initiated by the linear amplification of the target H1N1. The principle is as Figure 1 shown. The proposed SPA-LFA includes two steps: (1) Obtaining SPA products, including the isothermal amplification reaction of SPA for two partially complementary DNA fragments. The principle is as Figure 1 shown in A of. 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 at the 3′-end domain is complementary to the template probe Template and is used to initiate amplification; the fragment located at the 5′-end domain is used as the target recognition fragment and is complementary to a part of the H1N1 sequence. (2) Detecting the conjugate SPA of LFA. The principle is as Figure 1As shown in B of . First, Bio-11-UTP (U-Biotin) and the prepared SPA product ssDNA were added. In the presence of the target H1N1 RNA, a complex composed 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 was approximately 20 bp, with 10 bp complementary to the 3' end of the FAM probe and the other 10 bp complementary to the 5' end of the SPA-ssDNA. Finally, the formed "FAM-Biotin / target H1N1 / SPA product" ternary complex bound to the SA-AuNPs (nanogold labeled with streptavidin) on the test strip through the high recognition efficiency of streptavidin SA and biotin Biotin. Subsequently, the complex was detected by the anti-Fam monoclonal antibody embedded in the T line through immune recognition. 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 detecting the color depth of the test line T line. When the target H1N1 RNA was not present, the "FAM-Biotin / target H1N1 / SPA product" complex could not be formed, and no red band appeared on the T line. The whole operation process did not require magnetic separation and washing steps and a temperature-changing environment, and rapid detection visible to the naked eye could be achieved under normal temperature conditions.

[0037] II. The oligonucleotides used in this study are shown in Table 1.

[0038] Table 1

[0039]

[0040] III. Composition of the visual lateral flow analysis biosensor for influenza virus H1N1.

[0041] Primer Primer-9, Template Template-9, Bst DNA polymerase, dNTPs, PBS buffer, FAM Probe, LFA test strip.

[0042] IV. Obtaining biotin-labeled ssDNA by SPA isothermal amplification.

[0043] 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 Primer-9 (600 nM), 10 μL of template Template-9 (300 nM; the 3' end is 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, the reaction is carried out at 55 °C for 1 - 4 h, and the enzyme is inactivated at 80 °C. The biotin-labeled SPA product ssDNA is obtained. To improve the detection speed, ssDNA can be prepared in advance and stored at 4 °C for later use.

[0044] V. H1N1 detection method.

[0045] Download the gene sequence (CY147535.1) of influenza A virus (A / Puerto Rico / 8-SV14 / 1934(H1N1)) from the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ), and synthesize the RNA of H1N1 by Sangon Biotech. Mix 2 μL of FAM-Probe (1.5 μM), 5 μL of the SPA product ssDNA, and 28 μL of PBS buffer to form a 35-μL reaction system. Add the sample to be detected (RNA of H1N1) to the reaction system, and incubate at 37 °C for 10 min to form a FAM-Biotin biotin mixture. Drop the above mixture onto the LFA test strip, and continuously add 25 μL of PBS. The FAM-Biotin / target H1N1 / SPA product in the mixture binds to the colloidal gold nanoparticles and streptavidin (SA-AuNPs) on the conjugate pad of the LFA test strip and assembles under capillary action. Then, the FAM-Biotin / target H1N1 / SPA product is captured by the anti-Fam antibody immobilized on the T test line, resulting in the appearance of a red band. Make a preliminary evaluation with the naked eye. About 3 min after adding the sample, take an image with a smartphone and analyze and quantify it with ImageJ software.

[0046] VI. Statistical analysis.

[0047] Use ImageJ software to quantify the signal intensity of the nucleic acid test strip. The signal intensity is expressed as the T / C ratio, where T is the signal peak area of the test line and C is the signal peak area of the control line. Three parallel measurements are performed for each sample. The T / C values in the figure are expressed as the mean ± standard deviation (SD) of three parallel measurements. Use Prism 8 software for statistical analysis.

[0048] Example 2. Primer Screening and Feasibility Analysis.

[0049] I. Primer Screening.

[0050] To determine the most efficient primers in the biosensor (SPA-LFA), 4 pairs of primers and templates Primer-9 / Template-9, Primer-11 / Template-11, Primer-13 / Template-13, and Primer-15 / Template-15 were set. The numbers 9, 11, 13, and 15 represent the base numbers of the primers. SPA amplification was performed using the above primers respectively to obtain SPA products. The SPA products were characterized by atomic force microscopy, and the morphology of the SPA products was a typical DNA strand of 3.51 ± 0.04 nm (such as Figure 2 A and B), corresponding to the PAGE results in Figure 4 . According to the three-dimensional height profiles of the SPA products in A and B of Figure 3 , the 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, indicating that the SPA extension process can change the DNA sequence from the nanoscale to the microscale.

[0051] Polyacrylamide gel electrophoresis (PAGE) was performed on the SPA products, and imaging and analysis were carried out using ImageJ software to confirm the feasibility of the SPA isothermal amplification reaction and further demonstrate the amplification effect of SPA. First, the 4 pairs of primers and templates (5′ end) were labeled with fluorescent labels Cy5 and Cy3 respectively to verify the SPA efficiency. As Figure 4 shown, regardless of the primer length, SPA can successfully amplify when both the primer and the template are present, indicating the universality of the above 4 pairs of primers and templates. However, when using Primer-9 / Template-9, the amplification efficiency of the signal in lane 3 is relatively strong. Therefore, we selected Primer-9 / Template-9 as the reaction primer and template for subsequent experiments. Overall, the results proved the effectiveness of the SPA system.

[0052] II. Feasibility Analysis.

[0053] By designing deletion experiments on the components of the visual lateral flow analysis biosensor for influenza virus H1N1, the feasibility of the visual lateral flow analysis biosensor for influenza virus H1N1 was verified. The results are as Figure 5 shown in A and B. Only when all components are present, the peak T / C ratio will increase significantly. If one component is missing, it will result in no positive signal on the T line.

[0054] Example 3. Optimization of Reaction Conditions.

[0055] The amplification efficiency of the SPA isothermal amplification reaction is a key factor affecting the performance of the established biosensor. That is to say, the more ssDNA products of SPA are produced, the more binding sites there are, which can be more efficiently connected to the FAM-Probe probe, thereby improving the detection ability of the established biosensor.

[0056] Optimize the ratio of dTTP to UTP-Biorin. The results are as Figure 6 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 result in too few Bition sites on the ssDNA products of SPA, thus affecting the final binding efficiency with the test strip, while too high a concentration of UTP-Biotin will produce steric hindrance and reduce the amplification efficiency of SPA, resulting in a decrease in the amount of SPA product ssDNA.

[0057] Optimize the concentration of the FAM-Probe probe. Under the condition of the optimal ratio of dTTP to UTP-Biotin, optimize the concentration of the FAM-Probe probe. The SPA product ssDNA can provide binding sites for the FAM-Probe probe and the target H1N1 virus RNA, and the three can form a triple nucleic acid complex (FAM-Biotin / target H1N1 / SPA product), triggering the generation of a signal on the T line of the lateral flow assay test strip. However, an excessive amount of the 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, an excessive amount of free FAM-Probe probe will also cause unnecessary reagent waste and increase costs. Therefore, an appropriate concentration of the FAM-Probe probe can improve the detection performance of SPA-LFA. As Figure 6 shown in 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 is selected as the optimal concentration of the FAM-Probe probe.

[0058] Optimize the incubation time for detecting H1N1. Optimize the incubation time under the above optimal conditions. As Figure 6 shown in C, within the incubation time of 5 - 60 min, the performance of SPA-LFA does not change significantly. Therefore, 10 min is selected as the optimal incubation time in subsequent studies. The total time required for SPA-LFA detection is about 40 min, including 30 min for RNA extraction and 10 min for detection.

[0059] 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.

[0060] 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 ℃.

[0061] Example 4. Sensitivity analysis of the biosensor (SPA-LFA).

[0062] 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.

[0063] Table 2

[0064]

[0065] Example 5. Specific detection of biosensors.

[0066] To verify the specificity of the biosensor of the present invention for detecting HIN1 virus, RNase-free water was used as a negative control to detect the RNA of different subtypes of influenza viruses (H3N2, H5N1, H6N2, H7N9, and H9N2). The sample concentration was 1 × 10 5 pM to evaluate the specificity of SPA-LFA. As Figure 9 shown, SPA-LFA was positive only for HIN1 virus and 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 with other influenza virus subtypes (H3N2, H5N1, H6N2, H7N9, and H9N2) was also studied. As Figure 10 shown, there was almost no cross-reactivity between the target H1N1 virus and other subtypes, further confirming the excellent selectivity of SPA-LFA for HIN1 virus.

[0067] Example 6. Spike recovery experiment.

[0068] Spike recovery experiments were carried out 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 was affected by external interfering factors. First, 10% serum was treated with 0.2 U / μL of RNase inhibitor (to eliminate the influence on the target RNA). Subsequently, it was centrifuged at 6000 rpm for 20 minutes and then filtered through a 30 K ultrafiltration centrifugal tube to remove biomolecules. H1N1 RNA was diluted to 10 3 、10 4 and 10 5 pM with the treated 10% serum for recovery analysis. In this experiment, 10% biological serum without added H1N1 RNA was used as a negative control. At the same time, the same concentration of H1N1 RNA (10 3 、10 4 and 10 5 pM) was added to the saliva samples of healthy individuals to evaluate the recovery rate, and the saliva samples without added H1N1 RNA were used as negative controls.

[0069] Then, the biosensor of the present invention (SPA-LFA) 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 recoveries of saliva and serum samples were 93.8% - 130.7%, and the acceptable relative standard deviation was less than 11.1%. Generally speaking, these results confirmed the applicability of the biosensor of the present invention (SPA-LFA) for the detection of H1N1 virus in field and clinical settings.

[0070] Table 3

[0071]

[0072] Example 7. Verification of the detection effect of actual samples.

[0073] To evaluate the feasibility of the biosensor (SPA-LFA) for actual samples of the present invention, 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 a UV spectrophotometer and stored at -80 °C. The detected concentration was 1.0 × 10 4 ~ 1.0 × 10 6 copies / mL. Through naked-eye visual observation or grayscale analysis using ImageJ software, the results are as Figure 11 shown, and the practicability of the proposed biosensor (SPA-LFA) was explored. Compared with commercial influenza virus test strips, the target detected by commercial test strips is usually an antigen, which requires a long time of infection and a high concentration to determine a positive result. This method can directly detect H1N1 virus RNA. In the detection of RNA at different concentrations, H1N1 virus was accurately detected in all samples, and the visible signal intensity was obvious. In contrast, no signal was detected on the T line in the sample of allantoic fluid from uninfected chicken embryos.

[0074] Eight samples were simultaneously detected using a commercial gold-standard ELISA kit and the biosensor (SPA-LFA). Among them, samples 1 and 2 were allantoic fluid from the same negative non-infected chicken embryos at different times, and samples 3 - 8 were allantoic fluid from chicken embryos infected with different concentrations of H1N1. The results are shown in Table 4, indicating that the two methods showed consistency, further demonstrating the application ability of the biosensor (SPA-LFA) detection method in the detection of actual samples.

[0075] Table 4

[0076]

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 includes: Primer-H1N1, template Template-9, Bst DNA polymerase, dNTPs, PBS buffer, FAM Probe, LFA test strip; The sequence of the FAM Probe from 5' to 3' is FAM-GACAAGACCAATCCT; The sequence of Primer-H1N1 from 5' to 3' is GTCACCTCTGACTAA TCTCTTATT ; The sequence of Template-9 from 5' to 3' is AATAAGAGAAATAAGAGA-InvdT; The dNTPs are prepared by mixing dTTP and UTP-Biotin at a final concentration ratio of 1:

1.

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

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

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