LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit for double exotoxin genes of actinobacillus pleuropneumoniae

Abstract

The invention discloses a LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit for double exotoxin genes of actinobacillus pleuropneumoniae, and relates to the technical field of molecular biological detection. The kit comprises a primer combination, the primer combination comprises an LAMP (Loop-Mediated Isothermal Amplification) inner primer pair for detecting a target apxI gene and an LAMP inner primer pair for detecting a target apxIV gene, the LAMP inner primer pair for detecting the target apxI gene is composed of a primer ApxI-FIP and a primer ApxI-BIP, wherein the primer ApxI-FIP and the primer ApxI-BIP are used as primers; the LAMP inner primer pair used for detecting the target apxIV gene is composed of a primer ApxIV-FIP and a primer ApxIV-BIP. The kit can realize rapid detection and pathogenicity auxiliary analysis of actinobacillus pleuropneumoniae, and plays an important role in prevention and control of porcine contagious pleuropneumonia.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biological detection, in particular to a double exotoxin gene LAMP-LFD detection kit for Actinobacillus pleuropneumoniae. Background Art

[0002] Porcine pleuropneumonia is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae (APP), which can cause up to 10% mortality. At the same time, the disease increases the cost of treatment drugs, vaccines and other related expenses, affects feed conversion rate, and brings huge economic losses to the pig industry. At present, porcine contagious pleuropneumonia is endemic in most parts of the world. There are certain differences in the serotypes prevalent in different countries and regions. The European prevalent strain is serotype 2. Studies have shown that 90% of APP isolates in Denmark and Germany are serotype 2, and the prevalent APP isolates in the Netherlands are serotypes 9 and serotype 2. The prevalent strains of APP in the UK are serotypes 2, 3, 6, 7 and 8, the prevalent APP in North America is serotype 1 and serotype 5, and the prevalent APP strains in my country are serotypes 1, 3, 4, 5 and 7.

[0003] Studies have shown that ApxⅠ-Ⅲ and lipopolysaccharide (LPS) are important virulence factors of APP, which play a role in the formation of lung damage and can also stimulate the body to produce protective immunity. 19 serotypes of APP strains have been found. The virulence of different serotypes is related to the exotoxins produced by APP to a certain extent. Among them, ApxⅠ has strong hemolytic activity and strong cytotoxicity, ApxⅡ has weak hemolytic activity and moderate cytotoxicity, and ApxⅢ has no hemolytic activity and strong cytotoxicity. Generally, serotypes 1, 5, 9 and 11 are the most virulent, followed by serotypes 2 and 13, and serotypes 4, 6 and 7 are of medium virulence. Aerosol infection tests also show that serotypes 1, 5 and 9 are more virulent than serotypes 2, 6 and 7. Different serotypes of APP strains can only produce 1-2 of ApxⅠ-Ⅲ. Except for serotypes 10 and 14, other serotypes can produce ApxⅡ, while serotypes 1, 5, 9 and 11 can secrete ApxⅠ. ApxⅣ is only secreted after APP infects pigs, and is not secreted by APP cultured in vitro. At the same time, all serotypes of APP strains can produce it when infecting hosts, and it is often used as a diagnostic detection target. The clinical manifestations of porcine contagious pleuropneumonia include the most acute, acute and chronic infections. The most acute pigs may experience systemic shock and sudden death. Acute cases show dyspnea, fever and loss of appetite. Chronic cases have no obvious clinical manifestations. The pathogenic bacteria are stored in the crypts of the tonsils of pigs, making chronically infected pigs 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. Etiological diagnosis includes bacterial isolation, culture, strain identification, etc., but the bacterial isolation and identification process is long, and the separation success rate 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 microbial characteristic protein fingerprints. Compared with traditional microbial identification methods, it can complete the identification of microbial species at the species level within a few minutes, but this method is still limited by the acquisition of pure bacteria and requires the participation of large-scale instruments and equipment. Therefore, the development of sensitive and specific diagnostic technology for Actinobacillus pleuropneumoniae, especially on-site rapid detection technology that can detect highly pathogenic strains, has great application value for disease prevention and control.

[0004] Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method developed by NotoMi et al. It can amplify target DNA with high specificity and high efficiency under isothermal conditions. It does not require a high-precision PCR instrument, but only requires a simple heat preservation device (water bath) to complete the amplification of the target gene. This method uses a DNA polymerase with strand displacement activity and a set of four specially designed primers. The template, LAMP reaction buffer, etc. are added and placed at a certain temperature (60-68°C). It can be completed in one step. Its amplification efficiency is extremely high, and 10% of the DNA template can be achieved within 15-60 minutes. 9 ~10 10 Multiple amplification, so this method has the advantages of sensitivity, high efficiency, and low equipment dependence. Lateral flow dipstick (LFD) can realize LAMP amplification product analysis, with clear interpretation of results and convenient application. It also has the characteristics of multiple target analysis, which makes up for the single target analysis limitation of observing white magnesium pyrophosphate precipitation detection of LAMP reaction results. At present, there is no report on the dual LAMP-LFD detection of Actinobacillus pleuropneumoniae apxⅠ and apxⅣ. Therefore, the present invention intends to establish a dual LAMP-LFD detection method for Actinobacillus pleuropneumoniae apxⅠ and apxⅣ, in order to realize the rapid determination of whether the test sample contains Actinobacillus pleuropneumoniae, and analyze whether the sample contains apxⅠ at the same time, and suggestively interpret the pathogenicity of Actinobacillus pleuropneumoniae in the test sample, which has positive significance for the diagnosis, epidemiological investigation and prevention and control of porcine contagious pleuropneumoniae. Summary of the invention

[0005] The purpose of the present invention is to provide a double exotoxin gene LAMP-LFD detection kit for Actinobacillus pleuropneumoniae to solve the problems existing in the above-mentioned 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 porcine contagious pleuropneumoniae.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer combination for detecting Actinobacillus pleuropneumoniae based on dual LAMP-LFD, comprising a LAMP inner primer pair for detecting a target apxⅠ gene and a LAMP inner primer pair for detecting a target apxⅣ gene;

[0008] The LAMP inner primer pair for detecting the target apxⅠ gene consists of primers ApxⅠ-FIP and ApxⅠ-BIP;

[0009] The LAMP inner primer pair for detecting the target apxⅣ gene consists of primers ApxⅣ-FIP and ApxⅣ-BIP;

[0010] 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.

[0011] Furthermore, 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;

[0012] 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.

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

[0014] Furthermore, the product is a kit.

[0015] The present invention also provides a product for detecting Actinobacillus pleuropneumoniae based on double LAMP-LFD, comprising the above primer combination.

[0016] Furthermore, the product is a kit.

[0017] Furthermore, the kit also includes 2×BcaBEST Buffer, BcaBEST DNA polymerase, amplification product diluent, lateral flow dual nucleic acid detection test strips, positive controls and negative controls.

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

[0019] The present invention also provides a method for detecting toxin I and toxin IV of Actinobacillus pleuropneumoniae based on dual LAMP-LFD for non-disease diagnosis purposes, comprising the following steps:

[0020] Extracting genomic DNA of the sample to be tested;

[0021] Using the genomic DNA as a template, the above primer combination is used to perform LAMP amplification to obtain a LAMP amplification product;

[0022] After the LAMP amplification product is diluted, it is tested using a lateral flow dual nucleic acid test strip to observe and determine whether the test sample DNA contains toxin I and / or toxin IV.

[0023] Furthermore, the reaction system of the LAMP amplification is: 2×BcaBEST buffer 12.5 μL, BcaBEST 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;

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

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

[0026] The present invention develops a dual LAMP-LFD detection method and kit for Actinobacillus pleuropneumoniae, which 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 porcine contagious pleuropneumoniae.

[0027] The present invention uses only inner primers to perform LAMP amplification of apxⅠ and apxⅣ genes of Actinobacillus pleuropneumoniae, and no outer primers are involved in the reaction, thereby reducing potential interactions between primers (such as primer dimer formation), thereby improving the specificity of the reaction; in addition, the synthesis cost can be reduced, the experimental operation process is simplified, and the possibility of human error is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The figure is the result of the dual LAMP-LFD primer ratio optimization experiment; A is the electrophoresis result of the dual LAMP primer optimization; B is the test strip result of the dual LAMP primer optimization; in A, 1-3 and 5-7 are LAMP without external primers, wherein the ratio of ApxⅠ primer: ApxⅣ primer in 1-3 is 2.5:1.6, 2.5:2.0, 2.5:2.5, respectively, and the ratio of ApxⅠ primer: ApxⅣ primer in 5-7 is 3.0:1.6, 3.0:2.0, 3.0:2.4, respectively; 4 and 8 are LAMP under external primer conditions, and the ratio of ApxⅠ primer: ApxⅣ primer is 2.5:2.5, 3.0:2.4, respectively; M: Marker;

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

[0031] Figure 3 The result diagram of the dual LAMP-LFD reaction temperature optimization experiment; in A, 1-8 are LAMP reaction temperatures of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, and 9-10 are negative control LAMP reaction temperatures of 60°C and 67°C; M: Marker;

[0032] Figure 4 The result diagram of the sensitivity detection experiment of dual LAMP-LFD detection;

[0033] Figure 5 The results of the specific detection experiment of the dual LAMP-LFD detection are shown in FIG. 1 ; wherein SS2 represents Streptococcus suis type 2 ZY05719; HPS4 represents Glaseria parasuis type 4 SY16;

[0034] Figure 6 The results of common PCR test of clinical samples; 1-22: clinical samples; M: Marker; +: positive control; -: negative control;

[0035] Figure 7 The figure is the result of dual LAMP-LFD detection of clinical samples; A is the result of dual LAMP electrophoresis of clinical samples; B is the result of dual LAMP test strips of clinical samples; 1-22: clinical samples; M: Marker; +: positive control; -: negative control. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The operations in the following examples, unless otherwise specified, are all conventional experimental methods. The experimental materials in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0042] Actinobacillus pleuropneumoniae serotype 1 Shope strain, Streptococcus suis type 2 strain ZY05719, Glaseria parasuis type 4 SY16, and toxigenic Escherichia coli F5 were all supplied by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences.

[0043] APP serotype 1 Shope strain (published in the literature "Qiu Suoping, He Kongwang, Liu Zhongyong et al. Establishment and application of PCR rapid typing system for porcine actinobacillus pleuropneumoniae. Chinese Journal of Animal Quarantine, 2011, 28(8): 41-45") was streaked onto THB solid medium containing 0.01% (mass volume ratio) nicotinamide adenine dinucleotide (NAD), and cultured at 37°C for 16 h. 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), and divided into 2 portions, each of 50 μL. One portion was boiled in water for 10 min, centrifuged at 12000 rpm for 5 min, and the supernatant was collected as the APP serotype 1 strain template. The other portion was diluted 10 times with sterile PBS, and the appropriate dilution was selected for bacterial plate counting. The initial bacterial concentration was calculated to be 9.2×10 7 CFU / mL, from which it was deduced that 1 μL of the prepared APP serotype 1 strain template corresponds to 4.6×10 5CFU / μL.

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

[0045] 1. Design of LAMP Primer Set

[0046] The species-specific gene apxⅣ of Actinobacillus pleuropneumoniae was selected as the detection target gene. The ApxⅣ protein encoded by this gene is only secreted after APP infects pigs, but not secreted by APP cultured in vitro. At the same time, all serotypes of APP strains can produce it when infecting hosts. The apxⅠ gene encodes the ApxⅠ protein, which has strong hemolytic activity and strong cytotoxicity. Serotypes 1, 5, 9 and 11 strains can secrete ApxⅠ. At the same time, these serotype strains are generally highly pathogenic strains. Therefore, the apxⅠ gene is used as a suggestive detection target for highly pathogenic strains. According to the apxⅠ and apxⅣ genes, LAMP amplification primers were designed, and the inner primers were labeled respectively. The primer sequences are shown in Table 1.

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

[0048]

[0049] Note: Dig stands for digoxigenin group; Biotin stands for biotin group; 6-FAM stands for 6-carboxyfluorescein group.

[0050] 2. Optimization of LAMP primer concentration ratio, reaction temperature and reaction time

[0051] The single control variable method was used to optimize the primer concentration ratio, reaction temperature and reaction time of LAMP reaction. The concentration ratios of ApxⅠ primer: ApxⅣ primer were set to 2.5: (1.6, 2.0, 2.5) and 3.0: (1.6, 2.0, 2.4), respectively, and the reaction time was set to 20-60 min; the reaction temperature was set to 60-67 °C. After the reaction, the results were analyzed and verified by agarose gel electrophoresis or LFD test strips.

[0052] The results showed that when the ratio of ApxⅠ primer: ApxⅣ primer was 3.0:1.6, the LAMP amplification product showed clear and typical gradient bands ( Figure 1 A in the middle), and the characteristic apxⅠ amplification band will appear ( Figure 1 As shown by the arrow in A), the dual LFD test strip verifies that under this condition, two test lines will appear ( Figure 1 When the reaction time was 60 min, the electrophoresis bands of LAMP amplification products were clear, and there were characteristic apxⅠ amplification bands ( Figure 2As shown by the arrow in A), the dual LFD test strip verifies that under this condition, two test lines will appear ( Figure 2 When the reaction temperature is 60℃, the electrophoresis bands of LAMP amplification products are clear, and there are characteristic apxⅠ amplification bands ( Figure 3 (indicated by the arrow in the middle).

[0053] Figure 1 Lanes 4 and 8 of A also show the results of LAMP amplification under the conditions of external primers (see Table 2), with the concentration ratios of ApxⅠ primer:ApxⅣ primer being 2.5:2.5 and 3.0:2.4, respectively.

[0054] Table 2 Two pairs of LAMP primers for APP toxin gene amplification

[0055]

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

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

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

[0059] After the reaction is completed, take 5 μL of the LAMP amplification product, dilute it 20 times with ddH2O, take 100 μL of the diluted product, and drop it into the sample well of the lateral flow test strip. Observe the test strip results after 2-5 minutes. Result determination:

[0060] When bands appear in the quality control area (C), detection area 1 (T1) and detection area 2 (T2), the sample to be tested contains APP and APP contains the apxⅠ gene; when bands appear in the quality control area (C) and detection area 2 (T2), but no band appears in detection area 1 (T1), the sample to be tested contains APP but APP in the sample does not contain apxⅠ; when a band appears in the quality control area (C) but no band appears in detection area 1 (T1) and detection area 2 (T2), there is no APP in the sample to be tested; when no band appears in the quality control area (C), the test is deemed invalid regardless of whether there are bands in detection area 1 (T1) and detection area 2 (T2).

[0061] The lateral flow dual nucleic acid test strip (purchased from Wuhan Junno Biotechnology Co., Ltd., catalog number: A4622-50) consists of a sample pad, a conjugate pad, an NC membrane, and an absorption pad. The NC membrane detection zone 1 (T1) can capture the amplification product (red strip) of the apxⅠ internal primer pair (ApxⅠ-FIP, ApxⅠ-BIP); the NC membrane detection zone 2 (T2) can capture the amplification product (red strip) of the apx Ⅳ internal primer pair (ApxⅣ-FIP, ApxⅣ-BIP); the quality control zone (C) is to verify whether the sample volume and chromatography are normal. Regardless of whether the target gene exists in the sample, the quality control zone (C) will show a blue strip.

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

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

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

[0065] Dilute the prepared APP serotype 1 strain template to 10 3 CFU / μL, 10 2 CFU / μL, 10 CFU / μL, 5 CFU / μL, and the dual LAMP-LFD detection was performed using the dual LAMP-LFD kit of Example 2 to analyze the detection sensitivity. Figure 4 The results showed that the sensitivity of dual LAMP-LFD for detecting apxⅣ was 10 CFU, and the sensitivity of dual LAMP-LFD for detecting apxⅠ was 10 3 CFU.

[0066] Referring to the APP1 template preparation process, DNA templates of Streptococcus suis type 2 ZY05719, Glaseria parasuis type 4 SY16, and toxigenic Escherichia coli F5 were prepared respectively, and dual LAMP-LFD detection was performed using the dual LAMP-LFD kit of Example 2 to analyze the detection specificity. Figure 5 The results showed that there were no reactive bands in T1 and T2 for three other common swine infection bacteria.

[0067] Example 4 Clinical Application

[0068] Twenty-two pig lung samples from slaughterhouses were collected for LAMP-LFD detection. 0.5 g of lung tissue was cut aseptically, and 1 mL of THB liquid culture medium containing 0.01% (mass-volume ratio) nicotinamide adenine dinucleotide (NAD) was added. The culture was incubated at 37°C for 16 h, 500 μL of the enrichment solution was taken, centrifuged at 12,000 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, it was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected as the sample template.

[0069] Table 3 Common PCR primers

[0070]

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

[0072] Figure 6 The results showed that 22 samples were tested by conventional PCR, of which 2 samples were positive for apxⅣ gene, and all apxⅠ genes were negative, indicating that 2 of the 22 samples (No. 19 and 21) were positive for Actinobacillus pleuropneumoniae; Figure 7 The results in Figure A showed that 22 samples were tested by dual LAMP, of which 2 samples (No. 19 and 21) showed characteristic gradient bands, and the rest had no characteristic gradient bands. The two LAMP positive amplification products were subjected to LFD analysis. Figure 7 The results in middle B showed that both samples had bands at T2 and no bands at T1, indicating that both samples were positive for Actinobacillus pleuropneumoniae and negative for apxⅠ gene, which was consistent with ordinary PCR.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer combination for detecting Actinobacillus pleuropneumoniae based on dual LAMP-LFD, characterized in that: It includes a LAMP inner primer pair for detecting a target apxⅠ gene and a LAMP inner primer pair for detecting a target apxⅣ gene; The LAMP inner primer pair for detecting the target apxⅠ gene consists of primers ApxⅠ-FIP and ApxⅠ-BIP; The LAMP inner primer pair for detecting the target apxⅣ gene consists 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 as claimed in 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 of 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×BcaBEST Buffer, BcaBEST 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, and using the primer combination of claim 1 or 2 to perform LAMP amplification to obtain a LAMP amplification product; After the LAMP amplification product is diluted, it is tested using a lateral flow dual nucleic acid 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×BcaBEST buffer 12.5 μL, BcaBEST 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.

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