Swine transmissible gastroenteritis virus detection composition, method and kit based on RPA isothermal amplification and immunochromatography technology

Abstract

The invention discloses a swine transmissible gastroenteritis virus detection composition, method and kit based on RPA (recombinase polymerase amplification) isothermal amplification and immunochromatography technologies. The composition comprises an upstream primer, a downstream primer (with biotin modification) and a probe (5'end is modified into FAM, the 14th basic group C at the 3 'end is modified by THF, and the 3' end is provided with a polymerase extension blocking group C3-spacer), wherein the nucleotide sequences of the upstream primer, the downstream primer and the probe are shown as SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO.7. At the temperature of 37 DEG C, a large number of amplification products can be obtained after the kit reacts for 20 min, a visual result can be obtained on a test strip within 2 min, and the lowest detection limit is 8.2 * 10 <-2 > copies / microliter. A clinical sample detection result is consistent with that of RT-PCR, so that the kit is simple to operate, short in detection time, high in sensitivity and suitable for on-site rapid detection of the transmissible gastroenteritis virus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a porcine transmissible gastroenteritis virus detection composition, method and kit based on RPA isothermal amplification and immunochromatography technology. Background Art

[0002] Transmissible gastroenteritis (TGE) is an acute, highly contagious disease caused by the transmissible gastroenteritis virus (TGEV), which poses a serious threat to the healthy development of my country's pig industry. The clinical manifestations of the disease include watery diarrhea, vomiting, and dehydration. Pigs of all ages can be infected, especially piglets under 2 weeks of age, with a mortality rate of up to 100%. Recovered piglets often have problems with stunted development and growth retardation, and may evolve into "dead pigs". In recent years, TGE has often broken out simultaneously with porcine epidemic diarrhea, causing huge economic losses to the pig industry, making it an important diarrheal disease affecting the pig industry. There are currently no safe and effective drugs and vaccines, so rapid and accurate diagnosis is particularly important in the prevention and control of the disease.

[0003] Early detection and rapid diagnosis of epidemics are the key to scientific and effective prevention and control. However, existing molecular diagnostic techniques (such as RT-PCR and fluorescent quantitative PCR) and immunodiagnostic methods (such as ELISA) usually rely on expensive instruments and equipment, have a long detection time, and require professional personnel to operate and analyze. Relatively speaking, recombinase polymerase amplification (RPA), as an isothermal nucleic acid amplification technology, has a reaction temperature range of 37-42°C and can complete exponential amplification of target genes within 20-30 minutes. In addition, combining RPA with colloidal gold lateral flow assay (LFA) technology can not only further improve the specificity and sensitivity of the reaction, but also significantly reduce the dependence on high-end equipment, which makes this technology very suitable for rapid on-site diagnosis and meet the urgent needs of epidemic prevention and control. Summary of the invention

[0004] The purpose of the present invention is to provide a porcine transmissible gastroenteritis virus detection composition, method and kit based on RPA isothermal amplification and immunochromatography technology to solve the problems of expensive instruments and equipment, long detection time and the like in the prior art. The primer probe combination and kit provided by the present invention have strong detection specificity, higher sensitivity and accuracy, and can be effectively used for early rapid diagnosis of porcine transmissible gastroenteritis.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A porcine transmissible gastroenteritis virus detection composition based on RPA isothermal amplification and immunochromatography technology, comprising an RPA primer pair and a probe for RPA amplification, wherein the primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.6, and the probe comprises a probe primer having a nucleotide sequence as shown in SEQ ID NO.7

[0007] Furthermore, the 5' end of the downstream primer is modified with biotin, the 5' end of the probe is modified with FAM, the 14th base C from the 3' end of the probe is modified with tetrahydrofuran, and the 3' end of the probe has a polymerase extension blocking group C3-spacer.

[0008] The sequences of the primers and probes are listed in Table 1.

[0009] Table 1 Primer and probe sequences

[0010]

[0011] The present invention also provides a porcine transmissible gastroenteritis virus detection kit based on RPA isothermal amplification and immunochromatography technology, comprising a composition consisting of an RPA primer pair and a probe.

[0012] Furthermore, the kit also includes a porcine transmissible gastroenteritis virus positive plasmid standard, RPA enzyme lyophilized powder, a reaction buffer, sterile deionized water, a magnesium acetate solution and a test strip.

[0013] Furthermore, the positive plasmid standard is ZTOPO-TGEV, which is constructed by connecting the N gene of TEGV to the ZTOPO-Blunt / TA vector.

[0014] The present invention also provides a method for detecting porcine transmissible gastroenteritis virus based on RPA isothermal amplification and immunochromatography technology, comprising the following steps:

[0015] (1) extracting nucleic acid from the sample to be tested;

[0016] (2) performing RPA amplification using the primer pair and probe at a temperature of 35-42° C. for 10-45 minutes;

[0017] (3) Dilute the amplified product and detect it using a test strip to obtain and analyze the results.

[0018] Furthermore, the dilution step is to take 10 μL of amplification product and add 190 μL of sterile deionized water for dilution.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a new RPA primer pair and probe combination for porcine transmissible gastroenteritis virus (TGEV), which has the potential to be widely used in the rapid detection of porcine transmissible gastroenteritis virus. This method significantly shortens the detection time, and the RPA amplification time can reach 20 minutes. Even in the best case, the amplification process can be completed in only 10 minutes. This timeliness greatly enhances the ability to respond to epidemics in actual scenarios. In terms of temperature requirements, the method of the present invention also shows superiority, and can be effectively amplified within a temperature range of 30-42°C, while the ideal RPA amplification temperature is set at 37°C. This feature does not require complex constant temperature devices during on-site detection, and amplification can be achieved only by relying on higher ambient temperature or human body temperature, which is convenient for flexible application in different occasions. In addition, the kit prepared by the present invention exhibits extremely high sensitivity, with a minimum detection limit of up to 8.2×10 -2 copies / μL, which means that the occurrence of false negative results can be minimized, thereby improving the reliability of the operation. At the same time, the present invention has excellent specificity, which can ensure that there is no cross reaction with other porcine viruses, and effectively guarantee the accuracy of the test results. The test results of the present invention can also be easily judged by the naked eye without relying on any special equipment. This feature makes this method particularly suitable for rapid response on-site testing needs. The RPA amplification reaction in the entire detection process only takes 20 minutes, and then a 2-minute test strip test, which greatly shortens the overall time, bringing great convenience to on-site detection experiments.

[0021] The test results of the present invention on 50 pig farm clinical samples are completely consistent with the RT-PCR test results, proving the accuracy and reliability of the detection method provided by the present invention. In addition, the key to the success of RPA amplification lies in the design of primers. Minor primer differences may significantly affect the sensitivity and specificity of the detection. During the RPA amplification process, due to the possibility of non-specific binding between primers, false positive problems may occur. The present invention designs different primer pairs and performs systematic screening to optimize the combination of two pairs, and finally obtains the primer pairs with the strongest sensitivity and specificity. At the same time, by introducing the design of primer probes, not only the sensitivity and stability of RPA are further improved, but also the visualization analysis ability of the test results is enhanced. This makes it easier for users to understand the test results more intuitively, and improves the practicality and convenience of the application. In summary, the method of the present invention is suitable for on-site rapid detection of porcine transmissible gastroenteritis virus, and is simple to operate, has good specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of RPA primer screening for porcine transmissible gastroenteritis virus;

[0023] Figure 2 Temperature optimization results for RPA-LFD of porcine transmissible gastroenteritis virus;

[0024] Figure 3 Optimization results for RPA-LFD time for porcine transmissible gastroenteritis virus;

[0025] Figure 4 This is the sensitivity result of RPA-LFD detection of porcine transmissible gastroenteritis virus;

[0026] Figure 5 This is the sensitivity result of RPA-LFD test strip for porcine transmissible gastroenteritis virus;

[0027] Figure 6 It is the specific result of RPA-LFD test for porcine transmissible gastroenteritis virus;

[0028] Figure 7 It is the specific result of the RPA-LFD test strip for porcine transmissible gastroenteritis virus;

[0029] Figure 8 The results of RPA-LFD test for porcine transmissible gastroenteritis virus clinical samples;

[0030] Fig. 9 These are the results of RT-PCR testing of clinical samples for porcine transmissible gastroenteritis virus. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0032] Example 1 RT-PCR and RPA Primer Design for Porcine Transmissible Gastroenteritis Virus

[0033] The N gene sequences of multiple strains of TGEV viruses were downloaded from NCBI for comparative analysis. Specific RT-PCR primers and RPA primers were designed and synthesized within the conserved N gene sequence. When combined with the test strip, a Biotin group was modified at the 5' end of the downstream primer, a FAM group was labeled at the 5' end of the probe, a C3 Spacer group was labeled at the 3' end, and a THF group was labeled 14nt from the 3' end. The size of the RT-PCR amplification product is 386bp. The size of the RPA product is 143bp. Both primers and probes were synthesized by Sangon Biotech Co., Ltd.

[0034] Table 2 Primer and probe sequences of TGEV-N

[0035]

[0036]

[0037] Example 2 Screening of RPA Primers for Porcine Transmissible Gastroenteritis Virus

[0038] The designed 8 pairs of RPA primers were combined in pairs to obtain the following primer pairs: F1R1, F2R1, F3R1, F4R1, F1R2, F2R2, F3R2, F4R2, F1R3, F2R3, F3R3, F4R3, F1R4, F2R4, F3R4, F4R4, a total of 16 primer pairs. First, use sterile deionized water to dilute the primers to a working concentration of 10 μM, then prepare 50 μL RPA reaction system, and use the porcine transmissible gastroenteritis virus positive plasmid standard as a template. The specific steps are as follows:

[0039] 1. Add 10 μL of sterile deionized water and 29.5 μL of primer-free rehydration buffer to a sterilized 1.5 mL centrifuge tube.

[0040] 2. Add 2.4 μL of upstream and downstream primers of 16 pairs of primers and 3.2 μL of template respectively.

[0041] 3. Transfer the mixed solution in the centrifuge tube to the RPA enzyme lyophilized powder tube respectively, then add 2.5 μL 280 nM magnesium acetate solution to the tube cap, cover the tube cap, and vortex briefly until the lyophilized powder is completely dissolved.

[0042] Incubate at 4.37°C. 4 minutes after the reaction starts, mix by inverting the tube, then continue incubation for 16 minutes.

[0043] 5. Take 1 μL of RPA amplification product and dilute it with 4 μL PBS buffer, then add 1 μL loading buffer to the dilution, vortex to mix and centrifuge.

[0044] 6. Take 4 μL of the prepared product and perform electrophoresis on it using 3% agarose gel. The electrophoresis results are as follows: Figure 1 As shown, primers 1-16 are F1R1, F1R2, F1R3, F1R4, F2R1, F2R2, F2R3, F2R4, F3R1, F3R2, F3R3, F3R4, F4R1, F4R2, F4R3, and F4R4, respectively. The positive groups of the 16 primer pairs all amplified the target bands, among which the amplification product of primer pair 13 F4R1 was the best, so primer pair F4R1 was selected for subsequent experiments.

[0045] Example 3 Optimization of temperature conditions for porcine transmissible gastroenteritis virus RPA-LFD

[0046] According to the temperature range of RPA amplification reaction of 30-42°C, 6 different reaction temperatures of 30°C, 35°C, 37°C, 39°C, 40°C and 42°C were set for RPA amplification. The specific implementation steps are as follows:

[0047] 1. Prepare 50 μL RPA amplification system. The specific system is as shown in Example 2.

[0048] 2. Perform RPA amplification reaction, set 6 reaction temperatures at 30℃, 35℃, 37℃, 39℃, 40℃, and 42℃, and set up positive control group and negative control group. In the positive control group, add the positive plasmid standard of porcine transmissible gastroenteritis virus while keeping other conditions unchanged, and replace the positive plasmid standard of porcine transmissible gastroenteritis virus with sterile water in the negative control group. React under constant temperature, turn the reaction tube upside down several times after 4 minutes of reaction, and continue to react for 16 minutes, for a total of 20 minutes.

[0049] 3. Take 4 μL of the prepared products and perform electrophoresis on them using 3% agarose gel. Figure 2 As shown, No. 1-8 are the test results of samples corresponding to 30℃, 35℃, 37℃, 39℃, 40℃, 42℃, negative control and positive control respectively. The bands are very fuzzy at 35℃, and the brightness difference of the bands between 37℃ and 42℃ is not much. The middle 37℃ is selected as the optimal temperature for this test.

[0050] Example 4 Optimization of RPA-LFD time conditions for porcine transmissible gastroenteritis virus

[0051] According to the time range of RPA amplification reaction of 10-45min, 8 different reaction times of 10min, 15min, 20min, 25min, 30min, 35min, 40min, and 45min, 10 reactions of positive control and negative control were set for RPA amplification. The positive control group added the positive plasmid standard of porcine transmissible gastroenteritis virus under the same conditions, and the negative control group replaced the positive plasmid standard of porcine transmissible gastroenteritis virus with sterile water. RPA amplification was performed. The specific implementation steps are as follows:

[0052] 1. Prepare 50 μL RPA amplification system. The specific system is as shown in Example 2.

[0053] The RPA amplification reaction was carried out at a constant temperature of 2.37°C. 4 minutes after the reaction started, the 7 RPA reaction tubes were turned upside down, and the reaction was continued for 6 minutes, 11 minutes, 16 minutes, 21 minutes, 26 minutes, 31 minutes, 36 minutes and 41 minutes respectively.

[0054] 3. Take 4 μL of the prepared product and perform electrophoresis on it using 3% agarose gel. The results are as follows: Figure 3 As shown, No. 1-10 are the test results of samples corresponding to 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, negative control and positive control respectively. The target band can be observed after 10min of RPA amplification, and a brighter single band with a size of about 143bp appears at 20min. The brightness of the band is consistent from 20 to 45min. 20min is selected as the best time for this experiment.

[0055] Example 5 RPA-LFD sensitivity experiment of porcine transmissible gastroenteritis virus

[0056] To explore the sensitivity of the RPA-LFD method, the positive plasmid standard of porcine transmissible gastroenteritis virus was diluted in series from 8.2×10 6 -8.2×10 -3 copies / μL setting 8.2×10 6 copies / μL, 8.2×10 5 copies / μL, 8.2×10 4 copies / μL, 8.2×10 3 copies / μL, 8.2×10 2 copies / μL, 8.2×10 1 copies / μL, 8.2×10 0 copies / μL, 8.2×10 -1 copies / μL, 8.2×10 -2 copies / μL, 8.2×10 -3 The concentration gradient of 10 copies / μL was used as the template for detection. At the same time, a positive control group with the positive plasmid standard of porcine transmissible gastroenteritis virus as the template and a negative control with sterile water were set up. The specific implementation steps are as follows:

[0057] 1. Use sterile deionized water to dilute the template plasmid to 8.2×10 6 -8.2×10 -3 The concentration of the samples was adjusted to 10 copies / μL.

[0058] 2. Prepare 50μL RPA system. The specific system refers to Example 2.

[0059] 3. Perform RPA amplification reaction at 37°C for 20 min.

[0060] 4. Take 4 μL of the prepared product and perform electrophoresis on it using 3% agarose gel. The results are as follows: Figure 4As shown, No. 1-11 are 8.2×10 6 copies / μL, 8.2×10 5 copies / μL, 8.2×10 4 copies / μL, 8.2×10 3 copies / μL, 8.2×10 2 copies / μL, 8.2×10 1 copies / μL, 8.2×10 0 copies / μL, 8.2×10 -1 copies / μL, 8.2×10 -2 copies / μL, 8.2×10 -3 copies / μL, negative control and positive control corresponding to the test results of the sample, the template concentration is 8.2×10 6 -8.2×10 -3 In the 10 concentration gradient tests of 8.2×10 -3 No specific bands appeared in the 40 copies / μL, and the rest had bright single bands with a size of about 143bp.

[0061] 5. Combine lateral flow chromatography test strips to test the sensitivity of the RPA detection method. The red C line indicates that the detection is effective, and the red T line indicates that the sample test result is positive. After the RPA reaction is completed, take 10μL of nucleic acid amplification product and add 190μL of enzyme-free water to dilute the reaction solution, and take 50μL of the diluted reaction solution and drop it into the reaction well. Wait for 2-5 minutes to observe the results. The results are as follows Figure 5 As shown, No. 1-12 are 8.2×10 8 copies / μL, 8.2×10 7 copies / μL, 8.2×10 6 copies / μL, 8.2×10 5 copies / μL, 8.2×10 4 copies / μL, 8.2×10 3 copies / μL, 8.2×10 2 copies / μL, 8.2×10 1 copies / μL, 8.2×10 0 copies / μL, 8.2×10 -1 copies / μL, 8.2×10 -2 copies / μL, 8.2×10 - 3 The test results of the corresponding samples were 8.2×106 -8.2×10 -1 In the 8 concentration gradients of 10.2×10 copies / μL, red bands appeared in the quality control area (C line), and the bands in the detection area (T line) were all strong; at 8.2×10 -2 When the concentration of 1000 copies / μL was 2.34477 μg / mL, a red band appeared in the quality control area (line C), and the band in the detection area (line T) was weak; when the concentration of 1000 copies / μL was 2.3477 μg / mL, a red band appeared in the quality control area (line C), and the band in the detection area (line T) was weak; -3 When the number of copies / μL was 2.34, only the quality control area (line C) showed a red band, and the test area (line T) had no band. The results showed that the sensitivity of RPA-LFD reached 8.2×10 -2 copies / μL.

[0062] Example 6 Porcine transmissible gastroenteritis virus RPA-LFD specificity experiment

[0063] In order to explore the specificity of the RPA-LFD method, several common porcine viruses such as porcine transmissible gastroenteritis virus (TGEV), porcine blue ear virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV) were tested by RPA-LFD to verify the specificity of the present invention. At the same time, a positive plasmid standard of porcine transmissible gastroenteritis virus was used as a template as a positive control, and water was used as a template as a negative control.

[0064] The specific implementation steps are as follows:

[0065] 1. Virus nucleic acid extraction: Use a viral genomic DNA / RNA extraction kit to extract nucleic acids of porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, porcine deltacoronavirus, and porcine transmissible gastroenteritis virus and store them at -20°C.

[0066] 2. Prepare 50 μL RPA reaction system. The specific system is as shown in Example 2.

[0067] 3. Perform RPA amplification and react at 37°C for 20 min.

[0068] 4. Take 4 μL of the prepared product and perform electrophoresis on it using 3% agarose gel. The results are as follows: Figure 6 As shown, No. 1-5 are PRRSV, H 2 The test results of the samples corresponding to ZTOPO-TGEV, PEDV, PDCoV, and TGEV-N showed that only when the ZTOPO-TGEV positive plasmid standard was used as a template for the reaction, no bands were found when the porcine blue ear virus, porcine epidemic diarrhea virus, and porcine deltacoronavirus nucleic acid were used as templates. This indicates that the RPA method of the present invention has no cross-reaction with other porcine viruses and has good specificity.

[0069] 5. At the same time, the ZTOPO-TGEV positive plasmid standard, porcine transmissible gastroenteritis virus, porcine blue ear virus, porcine epidemic diarrhea virus, porcine delta coronavirus, and water were used for RPA reaction and combined with the test strips. The results are as follows: Figure 7 As shown, No. 1-6 are TGEV, TGEV-N, H 2 O, PRRSV, PEDV, and PDCoV test results, among which ZTOPO-TGEV positive plasmid standard and TGEV both showed two red lines, and the other three viruses and water only showed one red line at the C line position. This shows that the RPA-LFD method of the present invention has no cross-reaction with other porcine viruses and has good specificity for the detection of porcine transmissible gastroenteritis virus.

[0070] Example 7 Detection of clinical samples

[0071] Blood samples collected from pig farms were tested by RPA-LFD and compared with RT-PCR to evaluate the accuracy of the RPA-LFD method.

[0072] The specific implementation steps are as follows:

[0073] 1. Select 50 pig blood samples collected clinically in the pig farm, use a centrifuge, set the speed to 4000rpm, centrifuge for 5 minutes, separate the serum, and store it at 4℃ for later use.

[0074] 2. Extract nucleic acid according to the instructions of the commercial viral nucleic acid extraction kit and store at -20°C.

[0075] 3. Using the nucleic acids extracted from 50 serum samples as templates, RPA-LFD and RT-PCR methods were used for detection respectively, and the test results were compared to analyze the accuracy of RPA-LFD detection.

[0076] 4.RPA-LFD test results are as follows Figure 8 As shown in the figure, No. 1-6 are the RPA-LFD test results of TGEV positive samples. Among the 50 clinical samples, 6 of them had 2 red lines, and the rest had only 1 red line at the C line position, with a positive rate of 12%. 4 μL of the product prepared by RT-PCR method was taken and tested by 3% RT-PCR agarose gel electrophoresis. The results are shown in the figure. Fig. 9 As shown, No. 1-6 are the RT-PCR test results of TGEV positive samples. Among the 50 samples, 6 samples amplified specific bands, and the rest did not have any bands, and the positive rate was 12%. Therefore, the established RPA-LFD and RT-PCR test results are consistent. It shows that the RPA-LFD method of the present invention has good accuracy.

[0077] In summary, the porcine transmissible gastroenteritis virus visualization detection method provided by the present invention is suitable for rapid on-site detection, and has the advantages of simple operation, high sensitivity and strong specificity.

[0078] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0079] Although the embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A porcine transmissible gastroenteritis virus detection composition based on RPA isothermal amplification and immunochromatography technology, characterized in that: It comprises an RPA primer pair and a probe for RPA amplification, wherein the primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.6, and the probe comprises a probe primer having a nucleotide sequence as shown in SEQ ID NO.

7.

2. The composition according to claim 1, characterized in that The 5' end of the downstream primer is modified with biotin, the 5' end of the probe is modified with FAM, the 14th base C from the 3' end of the probe is modified with tetrahydrofuran, and the 3' end of the probe has a polymerase extension blocking group C3-spacer.

3. A porcine transmissible gastroenteritis virus detection kit based on RPA isothermal amplification and immunochromatography technology, characterized in that: The invention comprises the composition according to claim 1.

4. The kit according to claim 3, characterized in that The kit also includes a porcine transmissible gastroenteritis virus positive plasmid standard, RPA enzyme lyophilized powder, a reaction buffer, sterile deionized water, a magnesium acetate solution and a test strip.

5. The kit according to claim 3, characterized in that The porcine transmissible gastroenteritis virus positive plasmid standard is ZTOPO-TGEV, which is constructed by connecting the N gene of TEGV to the ZTOPO-Blunt / TA vector.

6. A method for detecting porcine transmissible gastroenteritis virus based on RPA isothermal amplification and immunochromatography technology, characterized in that: The following steps are involved: (1) extracting nucleic acid from the sample to be tested; (2) using the composition of claim 1 to perform RPA amplification at a temperature of 30-42° C. for 10-45 minutes; (3) Dilute the amplified product and detect it using a test strip to obtain and analyze the results.

7. The method according to claim 6, characterized in that The dilution step is to take 10 μL of amplification product and add 190 μL of sterile deionized water to dilute it.

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