CN110777220B - Primer group, probe, RPA test strip kit and identification method - Google Patents

Abstract

The invention belongs to the technical field of virus strain identification methods, and particularly relates to a primer set and a probe for detecting wild strains of African swine fever virus, which comprise a first primer set and a first probe; the upstream primer in the first primer group is shown as a sequence SEQ ID NO:1 is shown in the specification; the downstream primer in the first primer group is provided with biotin at the 5' end and has the sequence shown in SEQ ID NO:2, a DNA molecule shown in fig. 2; the first probe is formed by a sequence SEQ ID NO:3, and a DNA molecule shown in 3. Meanwhile, a primer group and a probe for detecting the MGF360-505R gene deletion strain are also provided; the primer group and the probe group have strong specificity and high sensitivity, and the invention also provides an RPA test strip kit and an identification method based on the RPA-LFD technology and the primer group and the probe group.

Description

A primer set, probe, RPA test strip kit, identification method

technical field

The invention relates to the technical field of virus strain identification methods, in particular to a primer set, a probe, an RPA test strip kit and an identification method.

Background technique

African swine fever (African swine fever, ASF) is an acute, highly contagious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), clinically characterized by high fever, loss of appetite, skin and Internal organ hemorrhage is the main feature, usually 2 to 10 days after the onset of death, and the mortality rate can reach 100% in severe cases. The disease belongs to the Class A animal disease that must be reported by the World Organization for Animal Health (OIE), and it is listed as a Class I animal disease in my country. African swine fever is endemic in dozens of countries in Africa, Europe and the Americas, causing huge economic losses and has spread to many Asian countries bordering Europe.

Since the first case of African swine fever broke out in my country in 2018, African swine fever has caused major losses to my country's pig industry and seriously affected the healthy development of my country's pig industry. Studies at home and abroad have shown that the knockout of the African swine fever virus MGF360-505R gene can significantly reduce the virus toxicity, and the live African swine fever virus with these two genes knocked out is expected to become a vaccine. At present, CN201910348878.7 and CN 201910700685.3 respectively disclose the gene-deleted attenuated African swine fever virus that can be used as a vaccine and the vaccine and its construction method. Through genetic engineering technology, the virulence gene of the African swine fever virus is deleted to obtain MGF360-505R deletion and A gene-deleted virus in which CD2V is co-deleted with MGF360-505R. Experiments have shown that the two strains can provide 100% immune protection against the virulent strains of African swine fever prevailing in China, and can be used as safe and effective vaccines for preventing and controlling the African swine fever epidemic in China, and have great social value. However, the use of the vaccine will lead to positive results of African swine fever virus detection by conventional methods, and it is impossible to distinguish the positive results from wild virus infection or vaccination.

At present, ASF diagnosis methods include direct immunofluorescence test, indirect immunofluorescence test, detection of viral nucleic acid and other methods. Among them, the nucleic acid detection method is widely used because of its simple operation and high sensitivity. Compared with these commonly used detection techniques, the recombinant enzyme polymerase amplification (RPA) technique can accurately detect ASFV in a shorter time. RPA technology mainly relies on three enzymes: recombinase, single-strand binding protein and DNA polymerase. The entire RPA reaction process is very fast, generally at a constant temperature of 37 ° C for 20 minutes to amplify a large number of target products. RPA reaction products can be quickly detected by flow immunoassay strips (LFD). RPA-LFD technology has the advantages of strong specificity, simple operation process, no initial heating step, high sensitivity, and rapid detection, which provides a possibility for the detection of African swine fever in the field.

Therefore, the technical problem to be solved in this application is: how to propose a primer set, probe, RPA test strip kit, and identification method based on RPA-LFD technology to identify African swine fever virus wild strains and MGF360-505R gene deletion strains. method.

Contents of the invention

The object of the present invention is to provide a kind of RPA detection primer group and probe group that distinguish African swine fever virus wild strain and MGF360-505R gene deletion strain, this primer group and probe group specificity are strong, sensitivity is high, simultaneously, this The invention also proposes an RPA test strip kit and identification method based on the RPA-LFD technology and the above-mentioned primer set and probe set.

Unless otherwise specified, % and parts in the present invention are percent by weight and parts by weight, and M represents mol/L.

To achieve the above object, the present invention provides the following technical solutions:

A primer set and probe for detecting African swine fever virus wild strain, including a first primer set and a first probe;

The upstream primer in the first primer set is shown in the sequence of SEQ ID NO: 1, which we named as: ASFV-MGF360-505R-F1;

ASFV-MGF360-505R-F1: 5'TGTGCTATTGCCCATAAGGATCTACATCTAT3' (SEQ ID NO: 1);

The downstream primer in the first primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 2; we named the downstream primer as: ASFV-MGF360-505R-R1;

ASFV-MGF360-505R-R1: 5' Biotin-CATACTCAGAATGCCTATTATATTTGTTGAATTG 3' (SEQ ID NO: 2);

The first probe is a DNA molecule with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end as shown in SEQ ID NO:3. We named the probe ASFV-MGF360-505R-P1;

ASFV-MGF360-505R-P1: 5'[FAM]TAACAGAATCGTACCCGATAAGTATCATCA[THF]TTTAGATATTCGCAT[C3-spacer]3'(SEQ ID NO:3);

Among them, biotin means biotin, FAM means carboxyfluorescein, THF means tetrahydrofuran linker, and C3-spacer means 3 methylene groups, which are used to prevent chain extension.

At the same time, the present invention provides a primer set and probe for detecting the MGF360-505R gene deletion strain, including a second primer set and a second probe;

The upstream primer in the second primer set is shown in the sequence of SEQ ID NO: 4; we named the upstream primer ASFV-MGF360-505RQS-F2;

ASFV-MGF360-505RQS-F2: 5' TATTTAATCATTTAGAGAAGGTCATCATAGGAG 3' (SEQ ID NO: 4);

The downstream primer in the second primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 5; we named the downstream primer ASFV-MGF360-505RQS-R2;

ASFV-MGF360-505RQS-R2: 5' Biotin-CAGGATACGATTCACTACAATAGTGAGTAC 3' (SEQ ID NO: 5);

The second probe is a DNA molecule with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end as shown in the sequence SEQ ID NO: 6; we named the second probe ASFV-MGF360 -505RQS-P2;

ASFV-MGF360-505RQS-P2:5'[FAM]TTCAACGAGCAGGAAACAACTGTGTGCTTA[THF]TACAGCAACATACCC[C3-space r]3' (SEQ ID NO: 6).

Among them, biotin means biotin, FAM means carboxyfluorescein, THF means tetrahydrofuran linker, and C3-spacer means 3 methylene groups, which are used to prevent chain extension.

Simultaneously, the present invention provides a kind of RPA test strip kit that distinguishes African swine fever virus wild strain and MGF360-505R gene deletion strain, and described kit contains:

a first primer set and a first probe, a second primer set and a second probe;

The upstream primer in the first primer set is shown in the sequence SEQ ID NO: 1;

The downstream primer in the first primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 2;

The first probe is a DNA molecule as shown in sequence SEQ ID NO: 3 with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end;

The upstream primer in the second primer set is shown in the sequence SEQ ID NO: 4;

The downstream primer in the second primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 5;

The second probe is a DNA molecule with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end as shown in SEQ ID NO:6.

In addition, this kit also includes: positive control substance and negative control substance;

Preferably, the positive control substance is a plasmid DNA containing the African swine fever virus MGF360-505R gene; the negative control substance is deionized water.

In the above RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains, DNA extraction reagents, RPA amplification reagents and lateral flow test strips are also included.

In the above RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains, magnesium acetate, ddH ₂ O, and 5×Extration Buffer are also included.

Finally, the present invention also provides a rapid method for distinguishing African swine fever virus wild strains and MGF360-505R gene deletion strains by RPA-LFD technology, comprising the following steps:

S1: extracting viral DNA from the sample;

S2: Put the extracted viral DNA into the RPA reaction system with the first primer set and the first probe, the second primer set and the second probe as described above to perform the RPA reaction, and obtain the amplification products A1 and A2 respectively

S3. Respectively fully react the amplified products A1 and A2 in step S2 with the 5×Extration Buffer to obtain products B1 and B2;

S4. The products B1 and B2 in step 3 are added dropwise to the sample holes (P1 and P2 test strips) of the lateral flow test strips described above, respectively, to carry out LFD reaction, observe the results, and determine the virus type.

Add the amplification products B1 and B2 respectively to the sample holes of the test strip, and the detection results are as follows:

1) When the detection area of the P1 test strip develops color, and the quality control area also develops color; the detection area of P2 develops color, and the quality control area also develops color, then the virus in the sample to be tested is a wild strain and MGF360-505R Mixed infection of gene deletion strains;

2) When the detection area of the P1 test strip develops color, and the quality control area also develops color; the detection area of P2 does not develop color, and the quality control area develops color, the virus in the sample to be tested is a wild strain infection;

3) When the detection area of P1 test strip does not develop color, and the quality control area develops color; the detection area of P2 develops color, and the quality control area also develops color, then the virus in the sample to be tested is the MGF360-505R gene deletion strain;

4) When the detection area of the P1 test strip does not develop color, and the quality control area develops color; the detection area of P2 does not develop color, and the quality control area develops color, then there is no virus in the sample to be tested.

5) When the quality control area does not develop color, the test strip is invalid, and a new test strip needs to be used for re-measurement.

In the above method, step S2 is specifically:

Take 2.1 μL each of the upstream primer and the downstream primer in the first primer set, the concentration of the upstream primer and the downstream primer is 10 μmol/L; 0.6 μL of the first probe with a concentration of 10 μmol/L, Rehydration Buffer 29.5 μL, ddH2O12 of RNase Inhibitor .2 μL, 1 μL of DNA template, mix and add to the RPA enzyme tube to shake and mix, then add 2.5 μL of magnesium acetate, and start the reaction to obtain the amplification product A1;

Take 2.1 μL each of the upstream primer and downstream primer in the second primer set, the concentration of the upstream primer and the downstream primer is 10 μmol/L; the second probe with a concentration of 10 μmol/L is 0.6 μL, Rehydration Buffer 29.5 μL, ddH2O12 of RNase Inhibitor .2 μL, 1 μL of DNA template, mixed and added to the RPA enzyme tube for shaking and mixing, then added 2.5 μL of magnesium acetate to start the reaction to obtain the amplification product A2.

In the above method, in the step S3, the volume of the amplified product A1 and the amplified product A2 is 10 μL; the volume of the 5×Extration Buffer is 80 μL.

In the above method, in the step S4, the volume of the products B1 and B2 is 75 μL, and the results are read at least 5 minutes after adding the sample into the sample well.

In the above method, in step S4, the RPA enzyme tube contains recombinase uvs X, single-strand binding protein and DNA polymerase; the RPA reaction conditions described in step S2 are: put the reaction system at 37°C React in a water bath for 20 minutes without high-temperature denaturation, annealing, and extension.

Compared with prior art, the beneficial effect of the present invention is:

1. Recombinase-polymerase amplification reaction (RPA), which uses specific primer pairs and probes to amplify the target fragment under constant temperature conditions. The whole process is carried out very quickly, and generally a detectable level of amplification product can be obtained within ten minutes. Flow immunoassay strip technology (LFD) is a test strip test for RPA amplification products, and the detection results of the amplification products on the lateral flow test strip can be observed within a few minutes, without complicated equipment, suitable for On-site rapid detection.

2. Through the ingenious design of the regions of primers and probes, this project can not only easily and quickly identify whether the tested samples are infected with African swine fever virus, and whether the infected strains have gene deletions, but also determine whether there are wild strains and genes The mixed infection of the deletion strain can be detected by test strips, and the results can be observed with the naked eye.

3. The present invention has the advantages of simple operation, strong specificity and high sensitivity, and only produces a specific amplification reaction to the DNA of African swine fever virus (ASFV), and it is not effective for swine fever virus (CSFV), porcine pseudorabies virus (PRV) , Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Parvovirus (PPV) nucleic acid and so on have no amplification reaction.

4. The present invention provides a new means for the detection of African swine fever virus in my country. The clinical application of the kit is convenient and practical, and it can be used for epidemic monitoring, differential diagnosis and purification of African swine fever virus in production practice. It can be used for the rapid identification of African swine fever virus strains in professional laboratories, and can provide technical support for improving the comprehensive prevention and control level of African swine fever in my country.

Description of drawings

Fig. 1 is the African swine fever virus (ASFV) RPA-LFD test result of embodiment 2.

Fig. 2 is the result of electrophoresis verification test of the African swine fever virus (ASFV) RPA product of embodiment 2.

Fig. 3 is the African swine fever virus (ASFV) RPA-LFD specific test result of embodiment 3.

Fig. 4 is the electrophoresis test result of the African swine fever virus (ASFV) RPA product specificity of embodiment 3.

Fig. 5 is the African swine fever virus (ASFV) RPA-LFD susceptibility test result of embodiment 4.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

Primer design

After a large number of comparisons of the 360-505R gene of the African swine fever virus MK333180.1 (ASFV) published in the GenBanK database of NCBI (National Center for Biological Information) and its front and rear parts are highly conserved and have specific regions, the design of ASFV 360- The 250 bp before and after the 505R gene and the ASFV 360-505R gene are specific primer pairs and probe sets.

Thus, a kind of RPA primer and probe for distinguishing ASFV wild-type strain and 360-505R gene deletion strain is provided, specifically:

Primer set and probe 1 for detection of wild strains:

Primer set 1:

ASFV-MGF360-505R-F1: 5'TGTGCTATTGCCCATAAGGATCTACATCTAT3' (SEQ ID NO: 1);

ASFV-MGF360-505R-R1: 5' Biotin-CATACTCAGAATGCCTATTATATTTGTTGAATTG 3' (SEQ ID NO: 2);

Probe 1:

ASFV-MGF360-505R-P1:5'[FAM]TAACAGAATCGTACCCGATAAGTATCATCA[THF]TTTAGATATTCGCAT[C3-spacer]3'(SEQ ID NO:3);

Primer set and probe 2 for detection of MGF360-505R gene deletion strain:

Primer set 2:

ASFV-MGF360-505RQS-F2: 5' TATTTAATCATTTAGAGAAGGTCATCATAGGAG 3' (SEQ ID NO: 4);

ASFV-MGF360-505RQS-R2: 5' Biotin-CAGGATACGATTCACTACAATAGTGAGTAC 3' (SEQ ID NO: 5);

Probe 2:

ASFV-MGF360-505RQS-P2:5'[FAM]TTCAACGAGCAGGAAACAACTGTGTGCTTA[TH F]TACAGCAACATACCC[C3-spacer]3' (SEQ ID NO: 6).

Among them, biotin means biotin, FAM means carboxyfluorescein, THF means tetrahydrofuran linker, and C3-spacer means 3 methylene groups, which are used to prevent chain extension.

The primer set and probe 1 amplified the 360-505R fragment of the ASFV wild strain by the RPA method, and the fragment lengths were 437bp and 381bp, respectively.

The primer set and probe 1 amplified the MGF360-505R gene deletion strain by RPA method, but the target fragment could not be amplified.

The primer set and probe 2 amplified the MGF360-505R gene deletion strain by RPA method, and the fragment lengths were 415bp and 263bp, respectively.

The primer set and probe 2 amplified the 360-505R gene of the ASFV wild strain by the RPA method, but the target fragment could not be amplified.

Example 2

RPA-LFD detection method

Materials and Methods

1.1 Primers in Example 1

1.2 Sample DNA extraction

There are no special requirements for DNA extraction, which can be extracted according to conventional methods or DNA extraction kits. The extracted DNA was stored at -20°C for later use or immediately used for RPA amplification.

1.3 Positive plasmid

The partial sequence of the missing 360-505R gene of ASFV published in the GenBanK database was artificially synthesized, connected to the pUC57 vector, transformed into E. coli competent cells DH5α, and spread on LB medium containing 50 mg/L kanamycin sulfate On the plate, culture at 37°C for 12-16 hours. After the bacteria were picked, screened and identified by sequencing, the plasmid was extracted after expanding the culture of positive bacteria, and the positive plasmid was named pUC57-360-505RQS.

After manual comparison of the published 360-505R gene and the partial sequences on both sides, primers ASFV-360-505R-F1/R1 were designed to amplify a sequence containing the 360-505R gene, ligated with pJET1.2 cloning vector, and transformed into large intestine Bacillus-competent cells DH5α were spread on LB medium plates containing 100mg/L ampicillin, cultured at 37°C for 12-16 hours. Designated pJET1.2-360-505R.

1.4 RPA-LFD experiment

Follow the steps below to conduct RPA-LFD experiment

S1. Extract viral nucleic acid from the sample according to the instructions of the nucleic acid extraction kit;

S2. With nucleic acid as template, carry out RPA reaction to sample respectively with primer set 1 and 2 and probe 1 and 2 described in embodiment 1, obtain amplified product; Adopt 50 μ L reaction system: upstream and downstream primer (10 μ mol/L ) each 2.1 μL, RPA probe (10 μmol/L) 0.6 μL, Rehydration Buffer 29.5 μL, RNase Inhibitor ddH2O 12.2 μL, DNA template 1 μL, after mixing, add to the RPA enzyme tube and shake to mix, then add 2.5 μL of magnesium acetate , the initial reaction. Wherein, the RPA enzyme tube contains recombinase uvs X, single-strand binding protein and DNA polymerase. The reaction condition of the RPA amplification reaction is: react in a heater at 37° C. for 20 min.

S3. Fully reacting the two products of the S2 amplification reaction with the 5×Extration Buffer. The reaction system includes: 10 μL of amplification product, 80 μL of 5×Extration Buffer, and fully mixed.

S4. Add the two products in S3 dropwise to the sample hole of the lateral flow test strip, observe the result, and determine the virus type. The reaction system is as follows: 75 μL is dropped into the reaction well of lateral flow test paper for LFD detection, and the result is read within 5 minutes.

The constructed positive plasmid samples were detected according to the above method. C represents the quality control band; T represents the test band. 1: primer set and probe 1+negative control; 2: primer set and probe 1+sample to be tested; 3: primer set and probe 2+sample to be tested; 4: primer set and probe 2+negative control.

Figure 1-(a) The sample to be tested is a wild strain without gene deletion;

Figure 1-(b) The sample to be tested is 360-505R gene deletion;

Figure 1-(c) The sample to be tested is a mixture of 360-505R gene deletion strain and wild strain.

The experimental results are shown in Figures 1-(a), 1-(b), and 1-(c).

1.5 Nucleic acid electrophoresis verification experiment of RPA reaction product

Follow the steps below to conduct RPA electrophoresis experiments

S1. The RPA amplification product of S2 in 1.4 was purified by adding phenol-chloroform solution (phenol:chloroform:isopropanol volume ratio 25:24:1) according to the volume ratio of 1:1, and centrifuged at a speed of 12000rmp/min for 8min. supernatant.

S2. Take 10 μL of the supernatant from step S1, add 1 μL of 10xloading buffer to mix, and then spot the sample on a 2% agarose gel well, electrophoresis in 1×TAE electrophoresis buffer at 120V, and observe the results with a gel imaging system.

The experimental results are shown in Figure 2; Note: M: DL2000 Maker; 1: Primer set and probe 1 + negative control; 2: Primer set and probe 1 + sample to be tested; 3: Primer set and probe 2 + sample to be tested ;4: primer set and probe 2 + negative control.

Figure 2-(a) The sample to be tested is a wild strain without gene deletion; Figure 2-(b) The sample to be tested is a 360-505R gene deletion; Figure 2-(c) The sample to be tested is a 360-505R gene deletion strain Mixed with wild strains.

The RPA electrophoresis verification results are consistent with the RPA-LFD experimental results.

Example 3

specificity test

To 1: 360-505R gene positive plasmid according to the RPA-LFD detection method that 1.4 establishes in embodiment 2; 2: swine fever virus (CSFV); 3: porcine pseudorabies virus (PRV); 4: pig reproductive and respiratory disorder synthesis 5: porcine parvovirus (PPV); 6: negative control (deionized water) for detection.

C represents the quality control band; T represents the test band. The results are shown in Figure 3.

The RPA product was identified according to the nucleic acid gel electrophoresis method established in 1.5 of Example 2. Note: 1: 360-505R gene positive plasmid; 2: CSFV; 3: Porcine pseudorabies virus (PRV); 4: Porcine reproductive and respiratory syndrome virus (PRRSV); 5: Porcine parvovirus ( PPV); 6: negative control (deionized water).

C represents the quality control band; T represents the detection band. The results are shown in Figure 4.

It can be seen from Figure 3 and Figure 4 that the corresponding 360-505R gene positive plasmid test strip is positive, and clear bands can be seen in the position of the corresponding fragment size in this swimming lane; while other pigs are common Infectious diseases such as 2: swine fever virus (CSFV); 3: porcine pseudorabies virus (PRV); 4: porcine reproductive and respiratory syndrome virus (PRRSV); 5: porcine parvovirus (PPV); and 6: negative The test strip corresponding to the control (deionized water) was negative, and no bands were shown in the corresponding swimming lanes. It shows that the detection method has good specificity.

Example 4

sensitivity test

Dilute the positive template pJET1.2-360-505R into 7 concentration gradients of 10 ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , and 10 ⁶ copies/μL.

The above-mentioned different template concentrations were detected according to the RPA-LFD method established in 1.4 of Example 2. The results are shown in Figure 5. As can be seen from accompanying drawing 5, the negative control (deionized water), the test strip of ^{10 0} only has the color development of the quality control band, and the detection band does not develop color ^; The control bands were all colored, indicating that the sensitivity of this method could reach 10 ¹ copies/μL.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

sequence listing

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Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd.

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Claims (3)

1. a kind of RPA test strip test kit that distinguishes African swine fever virus wild strain and MGF360-505R gene deletion strain, it is characterized in that, described test kit contains: the first primer set and the first probe, the second primer group and second probe; The upstream primer in the first primer set is shown in the sequence SEQ ID NO: 1; The downstream primer in the first primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 2; The first probe is a DNA molecule as shown in sequence SEQ ID NO: 3 with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end; The upstream primer in the second primer set is shown in the sequence SEQ ID NO: 4; The downstream primer in the second primer set is a DNA molecule with biotin at the 5' end as shown in the sequence SEQ ID NO: 5; The second probe is a DNA molecule with carboxyfluorescein at the 5' end and a C3-spacer group at the 3' end as shown in the sequence SEQ ID NO:6. 2. the RPA test strip kit of distinguishing African swine fever virus wild strain and MGF360-505R gene deletion strain according to claim 1, is characterized in that, also comprises DNA extraction reagent, RPA amplification reagent and lateral flow test strips. 3. The RPA test strip kit for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains according to claim 1, characterized in that it also includes magnesium acetate, ddH ₂ O, and 5×Extration Buffer.