LAMP primer composition, visualization kit, and applications for detecting feline panleukopenia virus.

Abstract

This invention relates to a LAMP primer composition, a visualization kit, and their applications for detecting feline panleukopenia virus, belonging to the field of molecular biology technology. It solves the technical problems of existing infectious disease detection methods, which are either low in sensitivity, high in cost, time-consuming, and unsuitable for on-site testing. The primer composition of this invention includes an outer primer, an inner primer, and a loop primer. The outer primer consists of an upstream primer F3 and a downstream primer B3, the inner primer consists of an upstream primer FIP and a downstream primer BIP, and the loop primer consists of an upstream primer LF and a downstream primer LB, with sequences shown in SEQ ID NO. 1-6, respectively. This primer composition has a significant advantage in reaction time and high specificity, which is more conducive to improving the accuracy of clinical diagnosis. Combined with a visualization kit consisting of lateral chromatography test strips, it does not require complex instruments and equipment, has a rapid reaction, and provides visualized results, making it more suitable for point-of-care testing.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a LAMP primer composition, visualization kit, and application for detecting feline panleukopenia virus, and particularly to the application of the primer composition in the preparation of LAMP products for detecting feline panleukopenia virus. Background Technology

[0002] Feline panleukopenia (FPV) is an acute, highly contagious feline disease caused by infection with feline panleukopenia virus (FPV). Symptoms primarily include vomiting, diarrhea, hemorrhagic enteritis, and leukopenia. It is one of the most important feline viral infectious diseases monitored in veterinary clinics. FPV mainly infects felines, with kittens aged 2-6 months being the most susceptible. After infection, FPV replicates primarily in tissues with the most active mitosis (such as the thymus, lymph nodes, intestinal crypts, and developing brain tissue), causing clinical symptoms such as vomiting, diarrhea, hemorrhagic enteritis, leukopenia, and ataxia. The mortality rate in kittens is as high as 85%. Currently, the global prevalence of FPV in pet cats is approximately 25%–80%, and in some densely populated cat-owning areas, the prevalence can reach over 95%. FPV is now widespread and prevalent worldwide, posing a significant threat to the health of pet cats due to its high infectivity, high morbidity, and high mortality rate. Furthermore, the frequent occurrence of FPV infections in tigers, lions, cheetahs, and even giant pandas poses a significant challenge to the protection of felines and other carnivores.

[0003] Currently, colloidal gold test strips, polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and nano-PCR detection technologies are widely used for the specific detection of FPV. While colloidal gold test strips are convenient and rapid, their sensitivity is relatively low. PCR, qPCR, and nano-PCR, although highly sensitive and specific, suffer from high costs and time consumption, making them unsuitable for primary-level veterinary hospitals and on-site testing. With the increasing number of pet cats, the market demand for point-of-care testing technologies suitable for pet cat trading, health checkups, and home diagnosis is becoming increasingly urgent. Therefore, a rapid, visual detection kit for FPV is urgently needed to provide a simple, economical, specific, and sensitive new detection method for veterinary hospitals with limited equipment and for on-site detection of FPV infection. Summary of the Invention

[0004] To address the technical problems of existing infectious disease detection methods, such as low sensitivity, high cost, long processing time, and unsuitability for on-site testing, this invention provides a LAMP primer composition, a visualization kit, and their applications for detecting feline panleukopenia virus.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0006] In a first aspect, the present invention provides a LAMP primer composition and visualization kit for detecting feline panleukopenia virus, comprising an outer primer, an inner primer, and a loop primer; The outer primer consists of an upstream primer F3 and a downstream primer B3. The sequence of the upstream primer F3 is shown in SEQ ID NO.1, and the sequence of the downstream primer B3 is shown in SEQ ID NO.2. The inner primer consists of an upstream primer FIP and a downstream primer BIP. The sequence of the upstream primer FIP is shown in SEQ ID NO.3, and the sequence of the downstream primer BIP is shown in SEQ ID NO.4. The loop primer consists of an upstream primer LF and a downstream primer LB. The sequence of the upstream primer LF is shown in SEQ ID NO.5, and the sequence of the downstream primer LB is shown in SEQ ID NO.6.

[0007] Preferably, the 5' end of the upstream primer FIP is labeled with biotin, and the 5' end of the upstream primer LF is labeled with a FAM group or FITC.

[0008] Secondly, the present invention provides the use of the above-mentioned primer composition in the preparation of LAMP products for detecting feline panleukopenia virus.

[0009] Preferably, the product is a reagent or a kit.

[0010] Thirdly, the present invention provides a LAMP visualization kit for detecting feline panleukopenia virus containing the above-mentioned primer composition, and further includes a lateral chromatography strip (LFD).

[0011] Preferably, it also includes LAMP reaction reagents.

[0012] More preferably, the LAMP reaction reagent is Bst II Pro DNA Polymerase or WarmStart® Multi-Purpose LAMP / RT-LAMP 2×Master Mix.

[0013] Preferably, the lateral chromatography test strip is a (general-purpose) nucleic acid test strip.

[0014] Preferably, the method further includes a positive plasmid of the FPV VP2 gene, which is prepared by the following method: using the DNA of the FPV isolate as a template, PCR amplification is performed using primers for the full-length FPV VP2 gene. After the amplification product is purified by gel recovery, it is cloned into the pGEM-T plasmid to obtain the positive plasmid of the FPV VP2 gene.

[0015] Fourthly, the present invention provides a non-diagnostic LAMP detection method for feline panleukopenia virus using the above-mentioned visualization kit, comprising the following steps: S1, Extract DNA from the sample to be tested; S2, LAMP amplification of the DNA of the sample to be tested was performed using a primer combination; S3. After diluting the amplification product 20-30 times with diluent, use a lateral chromatography test strip for detection and result interpretation. The result interpretation method is as follows: when the control line (C line) and the test line (T line) show color simultaneously, it indicates that the sample contains feline panleukopenia virus nucleic acid; when the control line (C line) shows color but the test line (T line) does not show color, it indicates that the sample does not contain feline panleukopenia virus nucleic acid; when the control line (C line) does not show color but the test line (T line) shows color, it indicates that the test is invalid and needs to be repeated.

[0016] Preferably, the LAMP amplification reaction process is as follows: reaction at 63~65℃ for 35~45 min.

[0017] Preferably, the LAMP amplification reaction system is 25 μL, comprising 12.5 μL LAMP reaction reagent, 2.5 μL 10× primer mixture, 2.0 μL template DNA, and 8.0 μL nuclease-free water; The LAMP reaction reagent is Bst II Pro DNA Polymerase or WarmStart® Multi-Purpose LAMP / RT-LAMP 2×Master Mix; The concentrations of upstream primer F3 and downstream primer B3 are each 2 μM, the concentrations of upstream primer FIP and downstream primer BIP are each 14 μM, and the concentrations of upstream primer LF and downstream primer LB are each 6 μM.

[0018] Preferably, the method for detecting the amplification product using a lateral chromatography test strip after diluting it 20-30 times with a diluent is as follows: take 5-10 μL of the amplification product and add it to nuclease-free water for a 20-30 fold dilution, then take 50 μL of the dilution and add it to the lateral chromatography test strip, and wait 5 minutes to observe the results.

[0019] Compared with the prior art, the present invention has the following technical effects: The LAMP primer composition of the present invention for detecting feline panleukopenia virus is designed based on the FPVVP2 gene in GenBank and includes an outer primer (F3 / B3), an inner primer (FIP / BIP), and a loop primer (LF / LB), which can specifically recognize eight independent regions on the FPV virus VP2 gene.

[0020] The LAMP primer composition for detecting feline panleukopenia virus of the present invention has significant advantages in reaction time, high specificity, and wide detection limit, which is conducive to improving the accuracy of clinical diagnosis. Combined with the visualization kit composed of lateral chromatography strips (LFD), it does not require complex instruments and equipment, has a rapid reaction and visualizes the results, and is more suitable for clinical point-of-care diagnosis in primary veterinary hospitals and field environments, with a wider range of application prospects.

[0021] The LAMP visualization kit for detecting feline panleukopenia virus of the present invention is simple and rapid to operate, does not rely on complex instruments and equipment, provides visualized results, and is suitable for clinical point-of-care testing of FPV. Attached Figure Description

[0022] Figure 1 This section describes the design and validation of the LAMP primer set used for FPV detection in Example 1. (A) shows the fluorescence amplification curves; red and pink represent two-repeat fluorescence amplification curves of the extracted FPV viral DNA template, while blue and light blue represent two-repeat fluorescence amplification curves of the nuclease-free water template. (B) shows the agarose gel electrophoresis results of the amplification products; "+" template represents FHV viral DNA, and "-" template represents nuclease-free water.

[0023] Figure 2 The graph shows the optimized reaction temperature results for the LAMP detection method of FPV in Example 2. In the graph, "+" represents the extracted FPV viral DNA, and "-" represents nuclease-free water.

[0024] Figure 3 The figure shows the optimization results of the inner primer concentration for the LAMP detection method of FPV in Example 2.

[0025] Figure 4 The figure shows the optimization results of the loop primer concentration for the LAMP detection method of FPV in Example 2.

[0026] Figure 5 The graph shows the reaction time optimization results of the LAMP detection method for FPV in Example 2. In the graph, "+" represents FPV viral DNA, and "-" represents nuclease-free water.

[0027] Figure 6This is a graph showing the specificity analysis results of the LAMP detection method for FPV in Example 3. In the graph, 1-7 represent, in order, positive plasmids of the FPVVP2 gene, extracted FPV viral DNA, FHV-1 isolates, FCV isolates, FCoV positive nucleic acid, Escherichia coli, and CPV isolates as templates, with NTC as a negative control.

[0028] Figure 7 This is a graph showing the sensitivity analysis results of the LAMP detection method for FPV in Example 4. The copy numbers of nucleic acids 1-9 are 1.0 × 10⁻⁹ CFU / mL, respectively. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 1.0×10 1 1.0×10 0 copies / μL, NTC is the negative control.

[0029] Figure 8 The graph shows the repeatability test results of the LAMP detection method for FPV in Example 5. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. The embodiments given below are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The use of terms as used in this specification is merely for describing particular embodiments and is not intended to limit the scope of this application.

[0032] The positive plasmid of the FPV VP2 gene used in the examples was prepared by the following method: using DNA from an FPV isolate (isolated, identified and preserved by the laboratory of Jilin Agricultural Science and Technology College, named CC-02 / 16) as a template, PCR amplification was performed using primers for the full-length FPV VP2 gene. After the amplification product was purified by gel extraction, it was cloned into the pGEM-T plasmid. The positive clone identified by PCR and sequencing was the positive plasmid of the FPV VP2 gene, named pGEM-VP2, and the sequence information can be found in GenBank (GenBank accession number: MF541121).

[0033] Example 1: LAMP primer set design and validation for feline panleukopenia virus Based on the conserved sequence of the FPV VP2 gene registered in GenBank, a LAMP primer set for specific detection of FPV was designed using Primer Explorer version 5 online software. This set includes outer primers (F3 / B3), inner primers (FIP / BIP), and loop primers (LF / LB), specifically recognizing eight independent regions on the FPV VP2 gene. The 5' end of the upstream primer FIP is labeled with biotin, and the 5' end of the upstream primer LF is labeled with a FAM group (or FITC). Primer sequences are shown in Table 1.

[0034] Table 1. LAMP primer set used for detecting feline panleukopenia virus.

[0035] Viral DNA was extracted from FPV isolate cell cultures (FPV isolates were isolated, identified and preserved by the laboratory of Jilin Agricultural Science and Technology College, and named CC-02 / 16 strain) using a viral DNA / RNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.).

[0036] The LAMP amplification system is shown in Table 2. The LAMP amplification reaction was performed in a LightCycler® 96 instrument under the following conditions: 65°C for 45 min, with fluorescence signals acquired every 30 s via the SYBR Green channel, followed by heating at 80°C for 5 min to terminate the reaction. The template was extracted FPV viral DNA.

[0037] LAMP amplification results are determined by the following methods: (1) fluorescence amplification curve generated by LightCycler® 96; (2) 2% agarose gel electrophoresis analysis.

[0038] Table 2 LAMP amplification reaction system

[0039] The results of fluorescence amplification curves and 2% agarose gel electrophoresis are as follows: Figure 1 As shown in the figure. The results show that the designed FPV LAMP primer set has a single fluorescence amplification curve, and the electrophoresis produces bright and clear ladder-like bands in the positive reaction, while the negative control has no band, indicating that the primer set can be used for the detection of FPV LAMP amplification.

[0040] Example 2: Establishment and Optimization of LAMP Detection Method for Feline Panleukopenia Virus The FPV was amplified using the LAMP primer set designed in Example 1, and the FPV viral DNA extracted in Example 1 was used as a template.

[0041] The amplification reaction volume was 25 μL, containing: 12.5 μL WarmStart Multi-Purpose LAMP / RT-LAMP 2×Master Mix, 2.5 μL 10× primer mixture (2 μM each of upstream primer F3 and downstream primer B3, 16 μM each of upstream primer FIP and downstream primer BIP, and 4 μM each of upstream primer LF and downstream primer LB), 2.0 μL template DNA, and 8.0 μL nuclease-free water. LAMP amplification conditions were: reaction at 65℃ for 60 min in a metal bath, followed by heating at 80℃ for 5 min to terminate the reaction. A positive plasmid of the FPV VP2 gene was used as a positive control, and nuclease-free water was used as a negative control.

[0042] After amplification, transfer 5 μL of the LAMP amplification product to a new nuclease-free centrifuge tube and dilute with 195 μL of nuclease-free water. Immerse the commercial LFD test strip in the dilution solution for 5–10 min. The results are interpreted as follows: the presence of only the control line indicates a negative result; the presence of both the control line and the test line indicates a positive result; and the absence of the control line indicates an invalid test.

[0043] 1. Optimization of reaction temperature The reaction was carried out according to the above reaction system, with reaction temperatures set at 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67°C, and reaction times of 60 min (other reaction systems and conditions remained unchanged). The reactions were conducted in metal baths with different temperatures. After the reactions were completed, commercially available LFD test paper was used for detection.

[0044] The results are as follows Figure 2 As shown, the reaction detection lines at 63℃, 64℃, and 65℃ exhibited the clearest color development and the highest grayscale value of the T line, with no significant difference among the three. Therefore, 63℃, 64℃, and 65℃ were determined to be the optimal reaction temperatures for the feline panleukopenia virus LAMP-LFD detection method.

[0045] 2. Optimization of internal primer concentration The reaction was carried out according to the above reaction system, with the outer primer concentration set at 0.2 μM, the loop primer concentration at 0.8 μM, and the inner primer concentrations set at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 μM, respectively. The reaction was conducted at a temperature of 64 °C for 60 min (other reaction systems and conditions remained unchanged) in a metal bath. After the reaction, detection was performed using commercially available LFD test strips.

[0046] The results are as follows Figure 3 As shown, when the inner / outer primer concentration ratio is 7:1 (both inner primers are 1.4 µM and both outer primers are 0.2 µM), the reaction detection line shows the clearest color development and the T line has the highest gray value. Therefore, the inner primer concentration is determined to be 1.4 µM.

[0047] 3. Optimization of Loop Primer Concentration The reaction was carried out according to the above reaction system, with the outer primer concentration set at 0.2 μM, the inner primer concentration at 1.4 μM, and the loop primer concentrations set at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 μM, respectively. The reaction was conducted at a temperature of 64 °C for 60 min (other reaction systems and conditions remained unchanged) in a metal bath. After the reaction, detection was performed using commercially available LFD test strips.

[0048] The results are as follows Figure 4 As shown, when the ratio of loop to outer primer concentration is 3:1 (both loop primers are 0.6 µM and both outer primers are 0.2 µM), the reaction detection line shows the clearest color and the T line has the highest gray value. Therefore, the inner primer concentration was determined to be 0.6 µM.

[0049] 4. Optimization of reaction time The reaction was carried out according to the above reaction system, using 10,000-fold diluted extracted FPV viral DNA as the template. The concentrations of the outer primers were all 0.2 μM, the inner primers were all 1.4 μM, and the loop primers were all 0.6 μM. The reaction temperature was set at 64℃, and the reaction time was 60 min, conducted in a metal bath. Reaction times were set at 10, 20, 30, 40, 50, and 60 min (other reaction systems and conditions remained unchanged). After the reaction, detection was performed using commercially available LFD test strips.

[0050] The results are as follows Figure 5 As shown, it was found that the band could be observed on the detection line after 20 minutes, and the band intensity reached a clear and stable level after 40 minutes. Therefore, 40 minutes was determined to be the optimal reaction time.

[0051] Example 3: Specificity analysis of the detection method for feline panleukopenia virus using LAMP LAMP amplification was performed using feline herpesvirus 1 (FHV-1) isolate CH-B, feline calicivirus (FCV) isolate CH-JL2, canine parvovirus (CPV) isolate BJ, positive nucleic acid of feline coronavirus (FCoV) (identified by quantitative real-time PCR), Escherichia coli (all identified and preserved in the laboratory of Jilin Agricultural Science and Technology College), extracted FPV viral DNA, and the FPV VP2 gene positive plasmid pGEM-VP2 as templates, according to the optimal reaction system and conditions determined in Example 2. After the reaction, commercially available LFD test strips were used for detection.

[0052] The results are as follows Figure 6 As shown, the results indicated that the extracted FPV viral DNA and the CPV isolate both showed bands on the detection line, consistent with the positive plasmid of the FPV VP2 gene; no reaction was observed with other pathogens or the negative control. This demonstrates that the LAMP detection method for FPV of the present invention has good specificity and no cross-reaction with other common feline pathogens, but it cannot distinguish between closely related FPV and CPV.

[0053] Example 4: Sensitivity analysis of the LAMP detection method for feline panleukopenia virus The positive plasmid of the FPV VP2 gene was serially diluted 10-fold to a nucleic acid copy number of 1.0 × 10⁻⁶. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 1.0×10 1 1.0×10 0 Copies / μL. Using the diluted FPV VP2 gene positive plasmid as a template, LAMP amplification was performed according to the optimal reaction system and conditions determined in Example 2. After the reaction, detection was performed using commercially available LFD test strips. Nuclease-free water was set up as a negative control.

[0054] The results are as follows Figure 7 As shown, the detection method of the present invention has a wide repeated detection range, capable of detecting 1.0 × 10⁻⁶. 8 ~1.0×10 2 The detection limit for nucleic acid is 100 copies / μL.

[0055] Example 5: Repeatability analysis of the detection method for feline panleukopenia virus using LAMP Using the 1.0 × 10 from Example 4 respectively 6 1.0×10 4 1.0×10 2 Positive plasmids of the FPV VP2 gene at a concentration of copies / μL were used as strong, moderate, and weak positive templates for LAMP amplification according to the optimal reaction system and conditions determined in Example 2. After the reaction, detection was performed using commercially available LFD test strips. Nuclease-free water was used as a negative control.

[0056] The results are as follows Figure 8 As shown, in three repeated tests, all positive reactions produced a visible red band at the T-line. T-line grayscale analysis revealed that the coefficients of variation for strongly positive and moderately positive samples were 7.32% and 6.48%, respectively, both below 10%. For weakly positive samples (i.e., the detection limit of this method), the coefficient of variation was 15.33%, exceeding 10%, but all three replicates consistently yielded positive results. This indicates that the LAMP detection method for FPV has good reproducibility.

[0057] Example 6: Clinical Sample Testing LAMP amplification was performed using the detection method described in Example 1 and the optimal reaction system and conditions determined in Example 2. The test samples were 53 fecal samples. 37 positive samples were detected, with a positive rate of 69.81%.

[0058] The commercially available FPV real-time PCR detection kit (Beijing Senkang Biotechnology Development Co., Ltd.) was used to detect 53 fecal samples, and 37 positive samples were detected. The results were consistent with those of the detection method of the present invention, indicating that the detection method of the present invention has a detection concordance rate of 100%, and that the two methods have a high degree of consistency.

[0059] In summary, this invention establishes a specific visual LAMP detection method for feline panleukopenia virus (FPV) targeting the VP2 gene. This method can amplify the FPV VP2 gene within 40 minutes at a constant temperature of 64°C, and the amplification can be visualized using lateral chromatography strips. The limit of detection for FPV1 nucleic acid standards is 10⁻⁶. 2 The repeatability was good. The overall concordance rate with qPCR for clinical samples was 100%. The FPV visualization LAMP detection method established in this invention is suitable for point-of-care clinical detection of feline panleukopenia.

[0060] The above description of the embodiments is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. All technical modifications made based on the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A LAMP primer composition for detecting feline panleukopenia virus, characterized in that, Including outer primers, inner primers, and loop primers; The outer primer consists of an upstream primer F3 and a downstream primer B3. The sequence of the upstream primer F3 is shown in SEQ ID NO.1, and the sequence of the downstream primer B3 is shown in SEQ ID NO.

2. The inner primer consists of an upstream primer FIP and a downstream primer BIP. The sequence of the upstream primer FIP is shown in SEQ ID NO.3, and the sequence of the downstream primer BIP is shown in SEQ ID NO.

4. The loop primer consists of an upstream primer LF and a downstream primer LB. The sequence of the upstream primer LF is shown in SEQ ID NO.5, and the sequence of the downstream primer LB is shown in SEQ ID NO.

6.

2. The LAMP primer composition for detecting feline panleukopenia virus according to claim 1, characterized in that, The upstream primer FIP is labeled with biotin at its 5' end, and the upstream primer LF is labeled with FAM or FITC at its 5' end.

3. The use of the primer composition according to claim 1 or 2 in the preparation of a LAMP product for detecting feline panleukopenia virus.

4. The application according to claim 3, characterized in that, The product is a reagent or kit.

5. A LAMP visualization kit for detecting feline panleukopenia virus containing the primer composition of claim 1 or 2, characterized in that, It also includes lateral chromatography test strips.

6. The visualization kit according to claim 5, characterized in that, It also includes one or two of the following: LAMP reaction reagent and positive plasmids of the FPV VP2 gene; The LAMP reaction reagent is Bst II Pro DNA Polymerase or WarmStart® Multi-PurposeLAMP / RT-LAMP 2× Master Mix; The positive plasmid of the FPV VP2 gene was prepared by the following method: using the DNA of the FPV isolate as a template, PCR amplification was performed using primers for the full-length FPVVP2 gene. After the amplification product was purified by gel recovery, it was cloned into the pGEM-T plasmid to obtain the positive plasmid of the FPV VP2 gene.

7. The visualization kit according to claim 5, characterized in that, The lateral chromatography test strip is a nucleic acid detection test strip.

8. A LAMP detection method for feline panleukopenia virus for non-diagnostic purposes containing the visualization kit of claim 5, characterized in that, Includes the following steps: S1, Extract DNA from the sample to be tested; S2, LAMP amplification of the DNA of the sample to be tested using the primer composition; S3. After diluting the amplification product 20-30 times with diluent, use a lateral chromatography test strip for detection and result interpretation. The result interpretation method is as follows: when the control line and the test line show color simultaneously, it indicates that the sample contains feline panleukopenia virus nucleic acid; when the control line shows color but the test line does not, it indicates that the sample does not contain feline panleukopenia virus nucleic acid; when the control line does not show color but the test line shows color, it indicates that the test is invalid and needs to be repeated.

9. The detection method according to claim 8, characterized in that, The LAMP amplification reaction process is as follows: reaction at 63~65℃ for 35~45 min; The LAMP amplification reaction system is 25 μL, including 12.5 μL LAMP reaction reagent, 2.5 μL 10× primer mixture, 2.0 μL template DNA and 8.0 μL nuclease-free water; The LAMP reaction reagent is Bst II Pro DNA Polymerase or WarmStart® Multi-Purpose LAMP / RT-LAMP 2×Master Mix; The concentrations of upstream primer F3 and downstream primer B3 are each 2 μM, the concentrations of upstream primer FIP and downstream primer BIP are each 14 μM, and the concentrations of upstream primer LF and downstream primer LB are each 6 μM.

10. The detection method according to claim 8, characterized in that, The method for detecting the amplified product using a lateral chromatography test strip after diluting it 20-30 times with a diluent is as follows: take 5-10 μL of the amplified product and add it to nuclease-free water for a 20-30 fold dilution, then take 50 μL of the diluted product and add it to the lateral chromatography test strip, and wait 5 minutes to observe the results.

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