A lamp-lfd detection kit for double exotoxin genes of actinobacillus pleuropneumoniae

Abstract

The application discloses a perfringens LAMP-LFD detection kit for double exotoxin genes of actinobacillus pleuropneumoniae, and relates to the technical field of molecular biology detection. The kit comprises a primer combination; the primer combination comprises a LAMP inner primer pair for detecting a target apx I gene and a LAMP inner primer pair for detecting a target apx IV gene; the LAMP inner primer pair for detecting the target apx I gene is composed of primers Apx I-FIP and Apx I-BIP; and the LAMP inner primer pair for detecting the target apx IV gene is composed of primers Apx IV-FIP and Apx IV-BIP. The kit can realize rapid detection of the actinobacillus pleuropneumoniae and auxiliary analysis of pathogenicity, and plays an important role in prevention and control of infectious pleuropneumonia of pigs.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular biological detection, in particular to a double-outer-toxin gene LAMP-LFD detection kit for Actinobacillus pleuropneumoniae. BACKGROUND

[0002] Porcine pleuropneumonia is a respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP) in pigs, which can cause a mortality rate of up to 10%, and the disease increases the cost of treatment drugs, vaccine immunization and other related expenses, affects the feed conversion rate, and brings huge economic losses to the pig industry. At present, porcine pleuropneumonia is endemic in most regions of the world, and the serotypes of the epidemic strains in different countries and regions are different. The epidemic strain in Europe is serotype 2, research shows that 90% of APP isolated strains in Denmark and Germany are serotype 2, the APP isolated strains prevalent in the Netherlands are serotype 9 and serotype 2, the APP epidemic strain in the United Kingdom is serotype 2, 3, 6, 7 and 8, the APP epidemic serotypes in North America are serotype 1 and serotype 5, and the APP epidemic strain in China is serotype 1, 3, 4, 5 and 7.

[0003] Research shows that ApxⅠ-Ⅲ and lipopolysaccharide (LPS) are important virulence factors of APP, which play a role in the formation of lung injury, and can also stimulate the body to produce protective immunity. It has been found that there are 19 serotypes of APP strains, and the virulence of different serotypes of strains is related to the production of exotoxin by APP to some extent. Among them, ApxⅠ has strong hemolytic activity and cytotoxicity, ApxⅡ has weak hemolytic activity and moderate cytotoxicity, and ApxⅢ has no hemolytic activity but strong cytotoxicity. Generally, serotypes 1, 5, 9 and 11 are the most virulent, serotypes 2 and 13 are less virulent, serotypes 4, 6 and 7 have moderate virulence, and aerosol infection test also shows that serotypes 1, 5 and 9 strains are more virulent than serotypes 2, 6 and 7. Different serotypes of APP strains can only produce 1-2 of ApxⅠ-Ⅲ, except serotypes 10 and 14, other serotype strains can produce ApxⅡ, and serotypes 1, 5, 9 and 11 strains can all secrete ApxⅠ. ApxⅣ is only secreted after APP infection in pigs, and APP cultured in vitro does not secrete it, and all serotypes of APP strains can produce it after infecting the host, which is commonly used as a diagnostic detection target. The clinical manifestations of porcine contagious pleuropneumonia include acute, acute and chronic infection. Acute cases can have systemic shock and sudden death, acute cases show dyspnea, fever and loss of appetite, and chronic cases have no obvious clinical manifestations. The pathogen is stored in the tonsil crypt of pigs, making chronic infected pigs become carriers and an important source of infection. The degree of lung lesions in APP infected pigs is closely related to the host, bacterial virulence, environment and feeding management. Bacteriological diagnosis includes bacterial isolation, culture, strain identification, etc., but the bacterial isolation and identification process is long, and the success rate of isolation is also limited by the culture conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) is a rapid identification technology based on the fingerprint of microbial characteristic proteins. Compared with traditional microbial identification methods, it can complete the identification of microbial species at the species level in a few minutes, but this method is still limited by the acquisition of pure bacteria, and large-scale instrument equipment is needed. Therefore, the development of sensitive and specific diagnostic techniques for Actinobacillus pleuropneumoniae, especially on-site rapid detection techniques that can indicate high virulence strains, has good application value for disease prevention and control.

[0004] Loop-mediated isothermal amplification (LAMP) technology is a nucleic acid amplification method developed by Noto Mi et al. It can amplify the target DNA under isothermal conditions with high specificity, high efficiency and rapidness, does not need to rely on high-precision PCR instruments, and only needs a simple incubation device (water bath) to complete the amplification of the target gene. The method uses a DNA polymerase with strand displacement activity and a set of four specially designed primers. After adding the template, LAMP reaction buffer and the like together at a certain temperature (60-68 DEG C), it can be completed in one step. The amplification efficiency is very high, and the DNA template can be amplified by 10 9 ~10 10 times within 15-60 min, so the method has the advantages of sensitivity, high efficiency and low dependence on equipment. Lateral flow dipstick (LFD) can realize LAMP amplification product analysis, the result is clear and convenient to apply, and has the characteristics of multiple target analysis, which makes up for the limitation of single target analysis of LAMP reaction result observation white magnesium pyrophosphate precipitate detection. At present, there is no report on the double LAMP-LFD detection of apx I and apx IV of actinobacillus pleuropneumoniae, therefore, the present application intends to establish a double LAMP-LFD detection method of apx I and apx IV of actinobacillus pleuropneumoniae, so as to realize the rapid determination of whether the detection sample contains actinobacillus pleuropneumoniae, analyze whether the sample contains apx I, prompt the pathogenicity of actinobacillus pleuropneumoniae in the detection sample, and have positive significance for the diagnosis, epidemiological investigation and prevention and control of swine infectious pleuropneumonia. SUMMARY

[0005] The purpose of the present application is to provide an actinobacillus pleuropneumoniae double exotoxin gene LAMP-LFD detection kit to solve the problems existing in the prior art. The kit can realize the rapid detection of actinobacillus pleuropneumoniae and auxiliary analysis of pathogenicity, and plays an important role in the immediate prevention and control of swine infectious pleuropneumonia.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides a primer combination for detecting actinobacillus pleuropneumoniae based on double LAMP-LFD, which comprises a LAMP inner primer pair for detecting target apx I gene and a LAMP inner primer pair for detecting target apx IV gene;

[0008] The LAMP inner primer pair for detecting target apx I gene is composed of primers Apx I-FIP and Apx I-BIP;

[0009] The LAMP inner primer pair for detecting the target apx IV gene is composed of primer Apx IV-FIP and Apx IV-BIP;

[0010] The nucleotide sequences of the Apx I-FIP, the Apx I-BIP, the Apx IV-FIP and the Apx IV-BIP are shown in SEQ ID NO. 1~4 respectively.

[0011] Further, the 5' end of the Apx I-FIP is labeled with a Digoxigenin group, and the 5' end of the Apx I-BIP is labeled with a Biotin group.

[0012] The 5' end of the Apx IV-FIP is labeled with a 6-FAM group, and the 5' end of the Apx IV-BIP is labeled with a Biotin group.

[0013] The application also provides application of the primer combination in preparation of a product for detecting Actinobacillus pleuropneumoniae based on double LAMP-LFD.

[0014] Further, the product is a kit.

[0015] The application also provides a product for detecting Actinobacillus pleuropneumoniae based on double LAMP-LFD, which comprises the primer combination.

[0016] Further, the product is a kit.

[0017] Further, the kit further comprises 2x BcaBEST Buffer, BcaBEST DNA polymerase, amplification product diluent, lateral flow double nucleic acid detection test strip, positive control and negative control.

[0018] Further, the positive control is genomic DNA of an Actinobacillus pleuropneumoniae serotype 1 strain.

[0019] The application also provides a method for detecting toxin I and toxin IV of Actinobacillus pleuropneumoniae based on double LAMP-LFD for non-disease diagnosis purposes, which comprises the following steps:

[0020] Genomic DNA of a sample to be detected is extracted;

[0021] The genomic DNA is used as a template, and the primer combination is used for LAMP amplification to obtain a LAMP amplification product;

[0022] After dilution of the LAMP amplification product, the lateral flow double nucleic acid detection test strip is used for detection, and whether toxin I and / or toxin IV is contained in the DNA of the detection sample is observed and determined.

[0023] Further, the reaction system of the LAMP amplification is: 2x BcaBEST buffer 12.5 μL, BcaBEST DNA polymerase 1.0 μL, primer Apx I-FIP 3.0 μL, primer Apx I-BIP 3.0 μL, primer Apx IV-FIP 1.6 μL, primer Apx IV-BIP 1.6 μL, template DNA 1.0 μL, ddH2O 1.3 μL;

[0024] The reaction procedure of the LAMP amplification is: 60℃ reaction for 60 min.

[0025] The present application discloses the following technical effects:

[0026] The present application develops a double LAMP-LFD detection method and kit for Actinobacillus pleuropneumoniae, which can realize rapid detection of Actinobacillus pleuropneumoniae and auxiliary analysis of pathogenicity, and plays an important role in instant prevention and control of porcine infectious pleuropneumonia.

[0027] The present application uses only internal primers for LAMP amplification of the apx I gene and the apx IV gene of Actinobacillus pleuropneumoniae, without participation of external primers, thereby reducing potential interaction (such as primer dimer formation) between primers, improving the specificity of the reaction, reducing the synthesis cost, and simplifying the experimental operation process and reducing the possibility of human error. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0029] Figure 1 Figure 1 is a result graph of double LAMP-LFD primer ratio optimization experiment; wherein, A is an electrophoresis result of double LAMP primer optimization; B is a test strip result of double LAMP primer optimization; in A, 1-3 and 5-7 are LAMPs without external primers, wherein Apx I primer:Apx IV primer in 1-3 are 2.5:1.6, 2.5:2.0, 2.5:2.5 respectively, and Apx I primer:Apx IV primer in 5-7 are 3.0:1.6, 3.0:2.0, 3.0:2.4 respectively; 4 and 8 are LAMPs with external primers, Apx I primer:Apx IV primer are 2.5:2.5, 3.0:2.4 respectively; M: Marker;

[0030] Figure 2Figure for results of time optimization experiment of double LAMP-LFD reaction; wherein, A is electrophoresis result of double LAMP primer optimization; B is test strip result of double LAMP primer optimization; in A, 1-6 are LAMP reaction for 20 min, 30 min, 40 min, 45 min, 50 min, 60 min respectively, 7 is negative control LAMP reaction for 60 min; M: Marker;

[0031] Figure 3 Figure for results of temperature optimization experiment of double LAMP-LFD reaction; wherein, in A, 1-8 are LAMP reaction temperature for 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃ respectively, 9-10 are negative control LAMP reaction temperature for 60℃, 67℃ respectively; M: Marker;

[0032] Figure 4 Figure for results of sensitivity detection experiment of double LAMP-LFD detection;

[0033] Figure 5 Figure for results of specificity detection experiment of double LAMP-LFD detection; wherein, SS2 represents Streptococcus suis type 2 ZY05719; HPS4 represents Parvovirus suis type 4 SY16;

[0034] Figure 6 Figure for results of ordinary PCR detection of clinical sample; 1-22: clinical sample; M: Marker; +: positive control; -: negative control;

[0035] Figure 7 Figure for results of double LAMP-LFD detection of clinical sample; wherein, A is electrophoresis result of double LAMP of clinical sample; B is test strip result of double LAMP of clinical sample; 1-22: clinical sample; M: Marker; +: positive control; -: negative control. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not intended to limit the present application, but to explain certain aspects, features, and embodiments of the present application.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, or any other stated value or implicitly supported value by every stated value or implicitly supported value within the stated range is expressly contemplated. The above description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

[0038] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would attribute to such terms. Although preferred methods and materials are described, any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0039] Many modifications and variations of the present disclosure described in the specification are possible without departing from the scope or spirit of the present disclosure. Other implementations of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. The specification and examples are illustrative only.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, as well as the possibility that one or more other elements can be added or otherwise included.

[0041] The operations in the following examples were carried out according to conventional experimental methods unless otherwise specified. The experimental materials used in the following examples were purchased from commercial channels unless otherwise specified.

[0042] APP serotype 1 Shope strain, Streptococcus suis type 2 ZY05719 strain, Paraglaserella multocida type 4 SY16, and toxin-producing Escherichia coli F5 were supplied by the Jiangsu Academy of Agricultural Sciences, Institute of Veterinary Medicine.

[0043] The APP serotype 1 Shope strain (which has been disclosed in the literature "Qiu Suoping, He Kongwang, Liu Zhongyong, et al. Establishment and application of PCR rapid typing system for Actinobacillus pleuropneumoniae. China Animal Quarantine, 2011, 28(8): 41-45") was streaked onto THB solid medium containing 0.01% (mass / volume) nicotinamide adenine dinucleotide (NAD) and incubated at 37°C for 16 hours. A single colony was picked and resuspended in 100 μL sterile PBS buffer (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl aqueous solution). The solution was divided into two parts, each of 50 μL. One part was boiled in a water bath for 10 minutes and then centrifuged at 12000 rpm for 5 minutes. The supernatant was collected as the APP serotype 1 strain template. The other part was diluted 10-fold with sterile PBS, and the appropriate dilution was selected for bacterial plate counting to calculate the initial concentration of the bacteria, which was 9.2 x 10 7 CFU / mL. Therefore, 1 μL of the prepared APP serotype 1 strain template corresponds to 4.6 x 10 5CFU / μL.

[0044] Example 1 Construction of a double LAMP-LFD detection method for Actinobacillus pleuropneumoniae exotoxin genes

[0045] 1. Design of LAMP primer sets

[0046] The apx IV gene, which is specific to the Actinobacillus pleuropneumoniae species and encodes the Apx IV protein that is only secreted after infection of pigs by APP, was selected as the target gene for detection. The Apx I protein, which is encoded by the apx I gene, has strong hemolytic activity and cytotoxicity, and is secreted by strains of serotypes 1, 5, 9, and 11. In addition, such strains are generally highly pathogenic. Therefore, the apx I gene was used as a target for the detection of highly pathogenic strains. LAMP amplification primers were designed based on the apx I and apx IV genes, and the inner primers were labeled separately. The primer sequences are shown in Table 1.

[0047] Table 1 LAMP amplification inner primers for two pairs of APP toxin genes

[0048]

[0049] Note: Dig represents a digoxigenin group; Biotin represents a biotin group; and 6-FAM represents a 6-carboxyfluorescein group.

[0050] 2. Optimization of LAMP primer concentration ratios, reaction temperatures, and reaction times

[0051] Single variable control method was used to optimize the LAMP reaction primer concentration ratios, reaction temperatures, and reaction times. The Apx I primer:Apx IV primer concentration ratios were set at 2.5:(1.6, 2.0, 2.5) and 3.0:(1.6, 2.0, 2.4), respectively, and the reaction times were set at 20-60 min. The reaction temperatures were set at 60-67°C. After the reaction, agarose gel electrophoresis or LFD test strips were used for analysis and verification.

[0052] The results showed that when the Apx I primer:Apx IV primer ratio was 3.0:1.6, the LAMP amplification product electrophoresis showed clear, typical gradient bands ( Figure 1 middle A), and a characteristic apx I amplification band ( Figure 1 indicated by the arrow in middle A) appeared. Double LFD test strips verified that two detection lines appeared under this condition ( Figure 1 middle B); when the reaction time was 60 min, the LAMP amplification product electrophoresis bands were clear, and a characteristic apx I amplification band ( Figure 2The double LFD test strip verified that two detection lines would appear under this condition (indicated by the arrows in Fig. 2B). Figure 2 Fig. 2B); when the reaction temperature was 60°C, the LAMP amplification product electrophoretic band was clear, and there was a characteristic apxI amplification band (indicated by the arrow in Fig. 2C). Figure 3

[0053] Figure 1 Lanes 4 and 8 of Fig. 2A also show the LAMP amplification results under the condition of outer primers (see Table 2), and the concentration of ApxI primer:ApxIV primer was 2.5:2.5 and 3.0:2.4, respectively.

[0054] Table 2 LAMP amplification outer primers for two pairs of APP toxin genes

[0055]

[0056] The final reaction system and reaction procedure were as follows:

[0057] The 25 μL double toxin LAMP reaction system of Actinobacillus pleuropneumoniae was prepared as follows: 2x BcaBEST buffer 12.5 μL, BcaBEST DNA polymerase 1.0 μL, ApxI-FIP primer 3.0 μL, ApxI-BIP primer 3.0 μL, ApxIV-FIP primer 1.6 μL, ApxIV-BIP primer 1.6 μL, template DNA 1.0 μL, and ddH2O 1.3 μL.

[0058] The LAMP amplification reaction procedure was as follows: 60°C for 60 min.

[0059] After the reaction, 5 μL of the LAMP amplification product was taken, diluted 20 times with ddH2O, 100 μL of the diluted product was taken, dropped into the sample well of the lateral flow test strip, and the test strip result was observed after 2-5 min. The result was determined as follows:

[0060] When the quality control zone (C), detection 1 zone (T1), and detection 2 zone (T2) all appeared bands, the sample contained APP, and the APP contained apxI gene; when the quality control zone (C) and detection 2 zone (T2) both appeared bands, and the detection 1 zone (T1) did not appear bands, the sample contained APP, but the APP in the sample did not contain apxI; when the quality control zone (C) appeared bands, and the detection 1 zone (T1) and detection 2 zone (T2) both did not appear bands, the sample did not contain APP; when the quality control zone (C) did not appear bands, regardless of whether the detection 1 zone (T1) and detection 2 zone (T2) had bands, the detection was considered invalid.

[0061] ​The lateral flow double nucleic acid detection test strip (purchased from Wuhan Junnuode Biotechnology Co., Ltd., item number: A4622-50) is composed of a sample pad, a conjugate pad, an NC membrane, and an absorption pad. The NC membrane detection 1 region (T1) can capture the amplification product (red band) of the apx I inner primer pair (Apx I-FIP, Apx I-BIP); the NC membrane detection 2 region (T2) can capture the amplification product (red band) of the apx IV inner primer pair (Apx IV-FIP, Apx IV-BIP); and the quality control region (C) is used to verify the sample amount and whether the chromatography is normal, and the quality control region (C) presents a blue band regardless of whether the target gene exists in the sample.

[0062] Example 2 Construction of double LAMP-LFD kit

[0063] The kit includes a double LAMP primer combination (SEQ ID NO. 1-SEQ ID NO. 4); 2x BcaBEST Buffer, BcaBEST DNA polymerase, amplification product diluent (ddH2O), lateral flow double nucleic acid detection test strip (same as in Example 1), positive control (genomic DNA of Actinobacillus pleuropneumoniae serotype 1 strain), and negative control (ddH2O).

[0064] Example 3 Sensitivity and specificity detection experiment of double LAMP-LFD kit

[0065] The prepared APP serotype 1 strain template was diluted to 10 3 CFU / μL, 10 2 CFU / μL, 5 CFU / μL, and the double LAMP-LFD kit of Example 2 was used for double LAMP-LFD detection to analyze the detection sensitivity. Figure 4 It is shown that the sensitivity of double LAMP-LFD detection of apx IV is 10 CFU, and the sensitivity of double LAMP-LFD detection of apx I is 10 3 CFU.

[0066] Referring to the APP1 template preparation process, DNA templates of Streptococcus suis type 2 ZY05719, Paraglaser porcine type 4 SY16, and toxin-producing Escherichia coli F5 were prepared, respectively, and the double LAMP-LFD kit of Example 2 was used for double LAMP-LFD detection to analyze the detection specificity. Figure 5 It is shown that the three other common pig infection bacteria have no reaction bands in T1 and T2.

[0067] Example 4 Clinical application

[0068] 22 samples of pig lung were collected from slaughterhouse for LAMP-LFD detection. 0.5 g of lung tissue was cut by sterile operation and added with 1 mL of THB liquid medium containing 0.01% (mass / volume) nicotinamide adenine dinucleotide (NAD), and then incubated at 37°C for 16 h. 500 μL of the incubated solution was centrifuged at 12000 rpm for 5 min, and the precipitate was resuspended in 100 μL of sterile PBS buffer. After boiling in a water bath for 10 min, the solution was centrifuged at 12000 rpm for 5 min, and the supernatant was collected as the sample template.

[0069] Table 3. Common PCR primers

[0070]

[0071] The 22 samples of lung were detected by common PCR (the primer sequences are shown in Table 3) and double LAMP-LFD method (using the double LAMP-LFD kit of Example 2) respectively.

[0072] Figure 6 The results showed that 2 samples were positive for apxIV gene and negative for apxI gene by common PCR detection of the 22 samples, indicating that 2 samples (No. 19 and 21) were positive for A. pleuropneumoniae. Figure 7 The results of double LAMP detection of the 22 samples showed that 2 samples (No. 19 and 21) had characteristic gradient bands, and the rest had no characteristic gradient bands. The LAMP positive amplification products of the 2 samples were analyzed by LFD, Figure 7 The results of double LAMP detection of the 22 samples showed that 2 samples (No. 19 and 21) had characteristic gradient bands, and the rest had no characteristic gradient bands. The LAMP positive amplification products of the 2 samples were analyzed by LFD,

[0073] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A primer combination for detecting Actinobacillus pleuropneumoniae based on dual LAMP-LFD, characterized in that: Used to detect targets apxⅠ LAMP primer pairs within genes and for detecting targets apxⅣ The composition of the primer pairs within the LAMP of the gene; The method for detecting the target apxⅠ The LAMP inner primer pair of the gene consisted of primers ApxⅠ-FIP and ApxⅠ-BIP; The method for detecting the target apxⅣ The LAMP internal primer pair for the gene consisted of primers ApxⅣ-FIP and ApxⅣ-BIP; The nucleotide sequences of the ApxⅠ-FIP, the ApxⅠ-BIP, the ApxⅣ-FIP and the ApxⅣ-BIP are shown in SEQ ID NOs. 1 to 4, respectively.

2. The primer combination according to claim 1, characterized in that The 5' end of the ApxⅠ-FIP is labeled with a Digoxigenin group, and the 5' end of the ApxⅠ-BIP is labeled with a Biotin group; The 5' end of the ApxIV-FIP is labeled with a 6-FAM group, and the 5' end of the ApxIV-BIP is labeled with a Biotin group.

3. Use of the primer combination according to claim 1 or 2 in preparing a product for detecting Actinobacillus pleuropneumoniae based on dual LAMP-LFD.

4. The use according to claim 3, characterized in that The product is a test kit.

5. A product for detecting Actinobacillus pleuropneumoniae based on dual LAMP-LFD, characterized in that: Comprising the primer combination according to claim 1 or 2.

6. The product according to claim 5, characterized in that The product is a test kit.

7. The product according to claim 6, characterized in that The kit also includes 2× Bca BEST Buffer, Bca BEST DNA polymerase, amplification product diluent, lateral flow dual nucleic acid detection test strips, positive control and negative control.

8. The product according to claim 7, characterized in that The positive control is genomic DNA of Actinobacillus pleuropneumoniae serotype 1 strain.

9. A method for detecting toxin I and toxin IV of Actinobacillus pleuropneumoniae based on dual LAMP-LFD for non-disease diagnosis purposes, characterized in that: The following steps are involved: Extracting genomic DNA of the sample to be tested; Using the genomic DNA as a template, LAMP amplification is performed using the primer combination of claim 1 or 2 to obtain a LAMP amplification product; After the LAMP amplification product is diluted, it is tested using a lateral flow dual nucleic acid detection test strip to observe and determine whether the test sample DNA contains toxin I and / or toxin IV.

10. The method according to claim 9, characterized in that The reaction system of the LAMP amplification is: 2× Bca BEST buffer 12.5 μL, Bca BEST DNA polymerase 1.0 μL, primer ApxⅠ-FIP 3.0 μL, primer ApxⅠ-BIP 3.0 μL, primer ApxⅣ-FIP 1.6 μL, primer ApxⅣ-BIP 1.6 μL, template DNA 1.0 μL, ddH2O 1.3 μL; The reaction procedure of the LAMP amplification is: reaction at 60° C. for 60 min.

Images