A microfluidic sample pretreatment chip for detecting different genotypes of african swine fever virus and a construction method and application thereof

Abstract

The application belongs to the technical field of biology and relates to a microfluidic sample pretreatment chip for detecting different genotype viruses of African swine fever and a construction method and application thereof. The application firstly provides a target gene SNP site combination for detecting African swine fever genotype I, II and I / II recombinant viruses; secondly, provides a LAMP system for specifically amplifying the target gene SNP site combination for detecting African swine fever genotype I, II and I / II recombinant viruses, and constructs a microfluidic sample pretreatment chip; finally, the sample for identifying the genotype of African swine fever virus is added into the microfluidic sample pretreatment chip for amplification, and the amplification product can be subjected to ASFV genotyping by detecting the above-mentioned SNP site combination on a genotyping dot blot chip, so that the detection, identification and monitoring of African swine fever genotype I, II and I / II recombinant viruses can be finally realized.

Description

TECHNICAL FIELD

[0001] The present technology belongs to the field of biotechnology, and specifically relates to a microfluidic sample pretreatment chip for detecting different genotypes of African swine fever virus and a construction method and application thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute, febrile, hemorrhagic, highly contagious infectious disease caused by African swine fever virus (ASFV). Different age groups and different breeds of pigs are susceptible, and the mortality rate is as high as 100%. It seriously endangers the global pig industry and brings huge economic losses to society. After infection with high virulence ASFV, the mortality rate of pigs is 100%, and after infection with moderate or low virulence ASFV, the mortality rate is less than 50%.

[0003] The ASFV genome is large (170Kb~190Kb) and very complex in structure, and there is no effective treatment method and commercial vaccine. The existing ASF prevention and control measures mainly include thorough disinfection of pig farms and surrounding environment, comprehensive culling of sick pigs, strict control of personnel and materials, and ASFV monitoring of pig herds. However, the intensity and effect of these prevention and control measures are still insufficient, especially in recent years, the genotype of the ASFV epidemic is becoming more and more complex, and the epidemic range of newly emerging virulent strains is continuously expanding, and the prevention and control of ASF is facing severe challenges. Therefore, it is urgent to research the means and methods for precise typing detection of ASFV and monitoring of ASFV epidemic and variation, to provide guidance for clinical practice and effectively prevent and control ASF.

[0004] Currently, the PCR and fluorescent PCR (qPCR) methods in the WOAH recommended method are the most widely used methods for detecting ASFV, but they cannot perform precise typing detection on the existing epidemic ASFV. Other ASFV detection methods mainly include virus isolation, red blood cell adsorption test (HA), fluorescent antibody test (FAT), double antibody sandwich ELISA test, indirect fluorescent antibody test (IFA), and direct immunofluorescence test (DIF), etc. These methods also cannot perform precise typing detection on the existing epidemic ASFV.

[0005] ASFV is divided into 24 genotypes according to the B646L gene.

[0006] Single nucleotide polymorphism (SNP) refers to the variation of single nucleotide occurring at the genome level, including single base conversion, transversion, insertion, deletion, etc., wherein part of the SNP sites has high conservation and high specificity, which can be used as a molecular marker for detecting viruses on a genotyping array chip. However, before the sample is detected by the genotyping array chip, a series of treatments are required for the virus, including extracting virus DNA, DNA quality inspection, DNA random amplification, DNA incubation, DNA fragmentation, DNA precipitation, DNA resuspension, etc., and overnight incubation for library construction is required, which is very time-consuming, usually more than 20 hours, the interference caused by non-specific amplification is strong, and nucleic acid contamination is easily produced.

[0007] Microfluidic chip technology is a frontier scientific technology that precisely manipulates fluid at the micron scale, and has the ability to integrate various operation units such as conventional biology or chemistry into a chip. Therefore, by designing different operation units on the microfluidic chip, the pre-treatment of DNA samples can be completed, especially the specific amplification of SNP sites, which greatly shortens the pre-treatment time of DNA samples and avoids non-specific amplification, which is of great significance for the detection and monitoring of viruses. At present, the ASFV detection method is a general type or single point detection, which cannot distinguish between ASFV genes I, II and I / II recombinant type. Therefore, a microfluidic sample pre-treatment chip capable of specifically amplifying the highly conserved SNPs sites within the type and highly specific SNPs sites between the types of ASFV genes I, II and I / II recombinant type is designed and prepared, which greatly shortens the DNA sample pre-treatment time and avoids non-specific amplification. By collecting the amplification product, accurate genotyping detection of ASFV on a genotyping array chip is realized, and the prevalence and variation of ASFV are monitored, which is of great significance for the prevention and control of ASF.

[0008] However, there is no microfluidic chip for specifically amplifying SNPs sites of major animal disease pathogens, and there is no method for specifically amplifying SNPs sites of ASFV genes I, II and I / II recombinant type for accurate genotyping detection. SUMMARY

[0009] In view of the above technical problems, the purpose of the present application is to design and prepare a microfluidic sample pre-treatment chip for specifically amplifying SNPs sites of target genes of ASFV genes I, II and I / II recombinant type, and to detect SNPs sites on a genotyping array chip for ASFV genotyping; for accurate detection of African swine fever virus genes I, II and I / II recombinant type, identification of recombinant genes of African swine fever virus genes I / II recombinant type, and monitoring of the prevalence and variation of African swine fever virus.

[0010] Specifically includes the following content:

[0011] In a first aspect, the present application provides a SNP site combination for detecting genotype I, II and I / II recombinant virus of African swine fever, which is composed of 16 SNP sites, and the 16 SNP sites are respectively:

[0012] SNP1: located at the 480th nucleotide of the B646L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with C / T polymorphism;

[0013] SNP2: located at the 600th nucleotide of the B646L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with G / A polymorphism;

[0014] SNP3: located at the 684th nucleotide of the B646L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with C / T polymorphism;

[0015] SNP4: located at the 711th nucleotide of the B646L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with T / C polymorphism;

[0016] SNP5: located at the 300th nucleotide of the CP204L gene CDS sequence of African swine fever virus with gene version number NC_044957.1, with A / T polymorphism;

[0017] SNP6: located at the 495th nucleotide of the CP204L gene CDS sequence of African swine fever virus with gene version number NC_044957.1, with C / T polymorphism;

[0018] SNP7: located at the 511th nucleotide of the CP204L gene CDS sequence of African swine fever virus with gene version number NC_044957.1, with C / T polymorphism;

[0019] SNP8: located at the 561st nucleotide of the CP204L gene CDS sequence of African swine fever virus with gene version number NC_044957.1, with C / T polymorphism;

[0020] SNP9: located at the 659th nucleotide of the MGF360-9L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with C / A polymorphism;

[0021] SNP10: located at the 717th nucleotide of the MGF360-9L gene CDS sequence of African swine fever virus with gene version number NC_044956.1, with T / C polymorphism;

[0022] SNP11: located at the 752th nucleotide of the CDS sequence of the MGF360-9L gene of African swine fever virus with gene version number NC_044956.1, with C / T polymorphism;

[0023] SNP12: located at the 873th nucleotide of the CDS sequence of the MGF360-9L gene of African swine fever virus with gene version number NC_044956.1, with C / T polymorphism;

[0024] SNP13: located at the 45th nucleotide of the CDS sequence of the MGF505-11L gene of African swine fever virus with gene version number NC_044956.1, with T / A polymorphism;

[0025] SNP14: located at the 150th nucleotide of the CDS sequence of the MGF505-11L gene of African swine fever virus with gene version number NC_044956.1, with A / C polymorphism;

[0026] SNP15: located at the 770th nucleotide of the CDS sequence of the MGF505-11L gene of African swine fever virus with gene version number NC_044956.1, with A / C polymorphism;

[0027] SNP16: located at the 879th nucleotide of the CDS sequence of the MGF505-11L gene of African swine fever virus with gene version number NC_044956.1, with A / G polymorphism.

[0028] In a second aspect, the application provides application of a reagent for detecting the SNP site combination of the first aspect in a chip / reagent kit for detecting African swine fever virus genotype I, II and I / II recombinant.

[0029] In a third aspect, the application provides application of a primer for specifically amplifying the SNP site combination of the first aspect in preparation of a microfluidic sample pretreatment reagent or chip for African swine fever virus genotype I, II and I / II recombinant.

[0030] In a fourth aspect, the application provides a LAMP reaction system set, which comprises a primer for specifically amplifying the SNP site combination of the first aspect.

[0031] Preferably, the LAMP reaction system set comprises LAMP reaction systems 1-16 for specifically amplifying SNP1-16 in the first aspect.

[0032] The LAMP reaction system 1 comprises: a forward external primer shown as SEQ ID NO. 1, a reverse external primer shown as SEQ ID NO. 2, a forward internal primer shown as SEQ ID NO. 3, and a reverse internal primer shown as SEQ ID NO. 4;

[0033] The LAMP reaction system 2 comprises: a forward external primer shown as SEQ ID NO. 5, a reverse external primer shown as SEQ ID NO. 6, a forward internal primer shown as SEQ ID NO. 7, and a reverse internal primer shown as SEQ ID NO. 8;

[0034] The LAMP reaction system 3 comprises: a forward external primer shown as SEQ ID NO. 9, a reverse external primer shown as SEQ ID NO. 10, a forward internal primer shown as SEQ ID NO. 11, and a reverse internal primer shown as SEQ ID NO. 12;

[0035] The LAMP reaction system 4 comprises: a forward external primer shown as SEQ ID NO. 13, a reverse external primer shown as SEQ ID NO. 14, a forward internal primer shown as SEQ ID NO. 15, and a reverse internal primer shown as SEQ ID NO. 16;

[0036] The LAMP reaction system 5 comprises: a forward external primer shown as SEQ ID NO. 17, a reverse external primer shown as SEQ ID NO. 18, a forward internal primer shown as SEQ ID NO. 19, and a reverse internal primer shown as SEQ ID NO. 20;

[0037] The LAMP reaction system 6 comprises: a forward external primer shown as SEQ ID NO. 21, a reverse external primer shown as SEQ ID NO. 22, a forward internal primer shown as SEQ ID NO. 23, and a reverse internal primer shown as SEQ ID NO. 24;

[0038] The LAMP reaction system 7 comprises: a forward external primer shown as SEQ ID NO. 25, a reverse external primer shown as SEQ ID NO. 26, a forward internal primer shown as SEQ ID NO. 27, and a reverse internal primer shown as SEQ ID NO. 28;

[0039] The LAMP reaction system 8 comprises: a forward external primer shown as SEQ ID NO. 29, a reverse external primer shown as SEQ ID NO. 30, a forward internal primer shown as SEQ ID NO. 31, and a reverse internal primer shown as SEQ ID NO. 32;

[0040] The LAMP reaction system 9 comprises: a forward external primer shown as SEQ ID NO. 33, a reverse external primer shown as SEQ ID NO. 34, a forward internal primer shown as SEQ ID NO. 35, and a reverse internal primer shown as SEQ ID NO. 36;

[0041] The LAMP reaction system 10 comprises: a forward external primer shown as SEQ ID NO. 37, a reverse external primer shown as SEQ ID NO. 38, a forward internal primer shown as SEQ ID NO. 39, and a reverse internal primer shown as SEQ ID NO. 40;

[0042] The LAMP reaction system 11 comprises: a forward external primer shown as SEQ ID NO. 41, a reverse external primer shown as SEQ ID NO. 42, a forward internal primer shown as SEQ ID NO. 43, and a reverse internal primer shown as SEQ ID NO. 44;

[0043] The LAMP reaction system 12 comprises: a forward external primer shown as SEQ ID NO. 45, a reverse external primer shown as SEQ ID NO. 46, a forward internal primer shown as SEQ ID NO. 47, and a reverse internal primer shown as SEQ ID NO. 48;

[0044] The LAMP reaction system 13 comprises: a forward external primer shown as SEQ ID NO. 49, a reverse external primer shown as SEQ ID NO. 50, a forward internal primer shown as SEQ ID NO. 51, and a reverse internal primer shown as SEQ ID NO. 52;

[0045] The LAMP reaction system 14 comprises: a forward external primer shown as SEQ ID NO. 53, a reverse external primer shown as SEQ ID NO. 54, a forward internal primer shown as SEQ ID NO. 55, and a reverse internal primer shown as SEQ ID NO. 56;

[0046] The LAMP reaction system 15 comprises: a forward external primer shown as SEQ ID NO. 57, a reverse external primer shown as SEQ ID NO. 58, a forward internal primer shown as SEQ ID NO. 59, and a reverse internal primer shown as SEQ ID NO. 60;

[0047] The LAMP reaction system 16 comprises: a forward external primer shown in SEQ ID NO. 61, a reverse external primer shown in SEQ ID NO. 62, a forward internal primer shown in SEQ ID NO. 63, and a reverse internal primer shown in SEQ ID NO. 64.

[0048] Preferably, the LAMP reaction system set further comprises a LAMP reaction system 17 for amplifying an internal reference gene, the LAMP reaction system 17 comprising: a forward external primer shown in SEQ ID NO. 65, a reverse external primer shown in SEQ ID NO. 66, a forward internal primer shown in SEQ ID NO. 67, and a reverse internal primer shown in SEQ ID NO. 68.

[0049] Preferably, the LAMP reaction systems 1-17 further comprise, respectively, a DNA polymerase, MgSO4, betaine, dNTPs, 75x SYBR Green Dye, and ddH2O.

[0050] Preferably, the LAMP reaction systems 1-17 respectively comprise: 0.1 μM of a forward external primer, 0.1 μM of a reverse external primer, 0.8 μM of a forward internal primer, 0.8 μM of a reverse internal primer, 2 U of HotStart Bst4.0 DNA polymerase, 20 mM of MgSO4, 2 μL of betaine, 3.5 mM of dNTPs, 75x SYBR Green Dye, and ddH2O supplemented to 20 μL.

[0051] Preferably, the LAMP reaction systems 1-17 are freeze-dried into microspheres, respectively.

[0052] In a fifth aspect, the present application provides a microfluidic sample pretreatment chip, wherein the microfluidic sample pretreatment chip is embedded with the LAMP reaction system set according to the fourth aspect.

[0053] Preferably, the microfluidic sample pretreatment chip is made of PDMS and is in the shape of a round cake and is composed of four layers, from top to bottom, a liquid flow channel layer, a PDMS elastic film layer, a gas flow channel layer, and a glass support layer.

[0054] The liquid flow channel layer is in the shape of a round cake, and a liquid outlet hole is arranged in the middle of the liquid flow channel layer; a peripheral liquid flow channel, 17 internal liquid flow channels, 17 amplification cavities and a product collection cavity are embedded in the bottom of the liquid flow channel layer but do not penetrate the top of the liquid flow channel layer; the peripheral liquid flow channel is arranged at the edge of the liquid flow channel layer, and a liquid sample hole is arranged on the peripheral liquid flow channel and penetrates the top of the liquid flow channel layer; the product collection cavity is arranged outside the liquid outlet hole; the 17 amplification cavities are arranged between the product collection cavity and the peripheral liquid flow channel and are distributed on the same circumference; the 17 internal liquid flow channels are arranged between the product collection cavity and the peripheral liquid flow channel, and respectively penetrate the 17 amplification cavities and are connected with the peripheral liquid flow channel and the product collection cavity; the peripheral liquid flow channel and the internal liquid flow channel realize sample flow, so that the sample enters the amplification cavity or collects sample amplification products; two air holes are arranged between the peripheral liquid flow channel and the amplification cavity, and the air holes penetrate the liquid flow channel layer and the PDMS elastic film layer and are embedded in but do not penetrate the gas flow channel layer.

[0055] The gas flow channel layer is in the shape of a round cake, and a gas flow channel 1 and a gas flow channel 2 are embedded in the bottom of the gas flow channel layer but do not penetrate the top of the gas flow channel layer; the position of the gas flow channel 1 corresponds to the middle of the product collection cavity and the amplification cavity of the liquid flow channel layer; the gas flow channel 2 penetrates the gas flow channel 1 and the two air holes; the air holes, the gas flow channel and the PDMS elastic film layer constitute a pneumatic valve of the microfluidic sample pretreatment chip; by controlling the gas flow, the pressure in the gas flow channel 1 is increased, the PDMS elastic film layer is stretched and tightly attached to the bottom of the internal liquid flow channel, the annular gas flow channel 1 intersects with the 17 internal liquid flow channels, and 17 pneumatic valves are formed, so that the 17 internal liquid flow channels are closed.

[0056] During detection, the LAMP reaction system for amplifying 16 SNP sites and one internal reference is embedded in the 17 amplification cavities; by controlling the gas nitrogen through the two air holes, the PDMS elastic film layer is stretched and tightly attached to the bottom of the internal liquid flow channel, so as to control the liquid flow and close the internal liquid flow channel; the amplification of 16 SNP sites and one internal reference in the sample DNA and the collection of the amplification products are completed; the liquid outlet hole is used to collect the amplification products of 16 SNP sites and one internal reference in the sample DNA for detection. The pneumatic valve structure is composed of the air holes, the gas flow channel and the PDMS elastic film layer; by introducing nitrogen through the gas flow channel, the stretched PDMS elastic film is tightly attached to the bottom of the liquid flow channel layer under the action of pressure, and the liquid flow is prevented.

[0057] In a sixth aspect, the LAMP reaction system group of the fourth aspect or the microfluidic sample pretreatment chip of the fifth aspect has any one of the following non-diagnostic purposes:

[0058] (1) Application in sample pretreatment for African swine fever virus detection and genotyping;

[0059] (2) Application in sample pretreatment for recombinant gene identification of African swine fever virus genotype I / II recombinant virus;

[0060] (3) Application in sample pretreatment for African swine fever virus epidemic and variation monitoring.

[0061] In a seventh aspect, the application provides a method for identifying African swine fever virus genotyping for non-diagnostic purposes, which comprises:

[0062] (1) extracting DNA from the sample to be tested;

[0063] (2) adding the sample DNA to the sample inlet of the microfluidic sample pretreatment chip of the fifth aspect, and closing the chip and reacting in a 65℃ water bath for 1h;

[0064] (3) collecting the amplification products of 16 SNP sites and 1 internal reference, and detecting on a genotyping array chip;

[0065] (4) when the detection results of SNP1-16 are C, G, C, T, A, C, C, C, C, T, C, C, T, A, A, A in sequence, the sample is determined to be ASFV genotype I; when the detection results of SNP1-16 are T, A, T, C, T, T, T, T, A, C, T, T, A, C, C, G in sequence, the sample is determined to be ASFV genotype II; and when the detection results of SNP1-16 are C, G, C, T, T, T, T, T, A, C, T, T, A, C, C, G in sequence, the sample is determined to be ASFV genotype I / II recombinant.

[0066] Compared with the prior art, the application has the following advantages and beneficial effects:

[0067] (1) The application provides a set of SNPs sites for identifying African swine fever virus genotype I, II and I / II recombinant virus, and designs primers for specifically amplifying the SNPs sites, and constructs a microfluidic sample pretreatment chip for specifically amplifying the SNPs sites, which significantly reduces non-specific amplification, avoids the interference of non-specific amplification on the detection results, and avoids false positive or false negative caused by non-specific amplification;

[0068] (2) The microfluidic sample pretreatment chip pre-embeds LAMP reaction system freeze-dried microspheres for specifically amplifying SNPs sites, which can greatly shorten the time length of DNA amplification and other pretreatment steps from more than 20 hours to less than 1.5 hours, and different operation units are designed in the microfluidic sample pretreatment chip, which reduces the sample usage and avoids nucleic acid pollution between samples through closed operation units;

[0069] (3) The microfluidic sample pretreatment chip is specially designed for sample pretreatment of African swine fever virus detection and typing, which can quickly, accurately, efficiently, specifically and automatically complete the pretreatment of DNA samples, and perform accurate detection and typing of ASFV on a genotyping dot array chip, so as to realize accurate detection and typing of ASFV, identification of recombinant genes of ASFV genotype I / II recombinant, and monitoring of ASFV epidemic and variation. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 The LAMP reaction system freeze-dried microspheres for specifically amplifying four target gene SNPs sites of ASFV genotype I, II and I / II recombinant type pre-embedded in the microfluidic sample pretreatment chip for specifically amplifying SNPs sites of African swine fever virus genotype I, II and I / II recombinant type;

[0071] Figure 2 The microfluidic sample pretreatment chip for specifically amplifying SNPs sites of African swine fever virus genotype I, II and I / II recombinant type is shown in the structural diagram; A is a perspective view, B is a liquid flow channel layer bottom view, C is a gas flow channel layer top view, and D is a gas valve working schematic diagram; wherein, 1 is a liquid flow channel layer, 2 is a PDMS elastic film layer, 3 is a gas flow channel layer, 4 is a glass support layer, 5 is a liquid sample hole, 6 is a liquid outlet hole, 7 is a gas hole, 8 is a peripheral liquid flow channel, 9 is an internal liquid flow channel, 10 is a product collection cavity, 11 is an amplification cavity, 12 is a gas flow channel 1, and 13 is a gas flow channel 2;

[0072] Figure 3 The microfluidic sample pretreatment chip for specifically amplifying SNPs sites of African swine fever virus genotype I, II and I / II recombinant type is shown in the structural diagram; A is a perspective view, B is a liquid flow channel layer bottom view, C is a gas flow channel layer top view, and D is a gas valve working schematic diagram; wherein, 1 is a liquid flow channel layer, 2 is a PDMS elastic film layer, 3 is a gas flow channel layer, 4 is a glass support layer, 5 is a liquid sample hole, 6 is a liquid outlet hole, 7 is a gas hole, 8 is a peripheral liquid flow channel, 9 is an internal liquid flow channel, 10 is a product collection cavity, 11 is an amplification cavity, 12 is a gas flow channel 1, and 13 is a gas flow channel 2;

[0073] Figure 4 The microfluidic sample pretreatment chip for specifically amplifying SNPs sites of African swine fever virus genotype I, II and I / II recombinant type is shown in the structural diagram; A is a perspective view, B is a liquid flow channel layer bottom view, C is a gas flow channel layer top view, and D is a gas valve working schematic diagram; wherein, 1 is a liquid flow channel layer, 2 is a PDMS elastic film layer, 3 is a gas flow channel layer, 4 is a glass support layer, 5 is a liquid sample hole, 6 is a liquid outlet hole, 7 is a gas hole, 8 is a peripheral liquid flow channel, 9 is an internal liquid flow channel, 10 is a product collection cavity, 11 is an amplification cavity, 12 is a gas flow channel 1, and 13 is a gas flow channel 2; Figure 1 ;

[0074] Figure 5 Results of detection of the microfluidic sample pretreatment chip for specific amplification of SNPs sites of African swine fever virus genotype I, II and I / II recombinant virus on the genotyping dot chip after sample pretreatment of ASFV Figure 2 ;

[0075] Figure 6 Results of detection of the microfluidic sample pretreatment chip for specific amplification of SNPs sites of African swine fever virus genotype I, II and I / II recombinant virus on the genotyping dot chip after sample pretreatment of ASFV Figure 3 . DETAILED DESCRIPTION

[0076] The present application will be further illustrated by the following detailed description of the specific embodiments. However, the following examples are only illustrative of the technical solutions of the present application and do not limit the technical solutions of the present application. Unless otherwise specified, the technical means used in the following examples are all conventional means known to those skilled in the art, the bioinformatics software and products used are all commercially available, and the experimental processes and methods are all conventional methods known in the art. The source of the materials used, trade name, and necessary composition are indicated at the first occurrence, and the same reagents are used thereafter unless otherwise specified.

[0077] In addition, it should be noted that the combination of sites and applications provided by the present application are achieved through the hard creative labor and optimization work of the inventors of the present application.

[0078] The SNP referred to in the present application refers to Single Nucleotide Polymorphism, which mainly refers to DNA sequence polymorphism caused by single nucleotide variation at the genomic level, and the single nucleotide variation includes variation caused by single base transition, transversion, insertion or deletion.

[0079] It should be noted that the molecular marker referred to in the present application is all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, molecular markers based on PCR technology, DNA markers based on restriction enzyme digestion and PCR technology, molecular markers based on DNA chip technology, and analysis marker technology based on EST database development. The molecular marker provided by the present application can be used for genome mapping and gene positioning research, map-based gene cloning, species relationship and system classification, etc.

[0080] It should be noted that the kit referred to in the present application is any box containing reagents used for detection or experiments, which is commonly used in the art to facilitate the operator to get rid of the tedious process of reagent preparation and optimization. In an embodiment of the present application, the kit contains primers for amplifying the site information provided by the present application, molecular markers or probes or gene chips for detecting the site information provided by the present application, and further contains enzymes and buffers used for amplification, or further contains fluorescent labels used for detection.

[0081] Example 1 Preparation method of microfluidic sample pretreatment chip for specifically amplifying SNPs sites of African swine fever virus genotype I, II and I / II recombinant virus

[0082] 1. Screening and identification of target genes for detecting African swine fever virus genotype I, II and I / II recombinant virus

[0083] As reported in the literature (Zhao D, Sun E, Huang L, et al. Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs[J]. Nat Commun.2023, 14(1): 3096-3106.), the whole genome sequence of ASFV genotype I / II recombinant is produced by recombination of part of the gene sequences of ASFV genotype I and ASFV genotype II, wherein the ASFV genotype I gene fragment accounts for 43.5%, and the ASFV genotype II gene fragment accounts for 56.5%. The B646L gene is an internationally recognized gene for detecting ASFV, which has undergone recombination in the ASFV genotype I / II recombinant, and the recombinant B646L gene belongs to the ASFV genotype I. Based on this, the B646L gene is determined as the first detection target gene. According to the variation and prevalence of ASFV, the CP204L gene, the MGF360-9L gene and the MGF505-11L gene are highly conserved genes with little variation in the whole genome of ASFV, therefore, the B646L gene, the CP204L gene, the MGF360-9L gene and the MGF505-11L gene are selected for bioinformatics analysis, sequence comparison, verification of the recombination of the four genes in the ASFV genotype I / II recombinant, and verification of whether the four genes can be used as the final detection target gene. The specific steps are as follows:

[0084] In the NCBI database, search "African swine fever virus", 348 ASFV full genome information can be retrieved. After sorting according to "Release Date", view the full genome information of the 348 ASFVs in chronological order, select the B646L gene, CP204L gene, MGF360-9L gene, and MGF505-11L gene in each ASFV full genome sequence, download the FASTA file of the CDS sequence, and after quality control, use jalviewg software for sequence comparison. The sequence alignment results of the four target genes found that the B646L gene, CP204L gene, MGF360-9L gene, and MGF505-11L gene are highly conserved in ASFV gene type I, type II, and I / II recombinant type, and highly specific SNPs sites are identified in different types of ASFV. In addition, the sequence alignment results found that in the ASFV gene I / II recombinant type, the B646L gene belongs to the ASFV gene type I, the CP204L gene, the MGF360-9L gene, and the MGF505-11L gene belong to the ASFV gene type II, all with 100% sequence identity. Sequence alignment of the four target genes of ASFV gene type I and ASFV gene type II found that the B646L gene of ASFV gene type I and ASFV gene type II is highly conserved, and 30 specific SNPs sites are identified; the CP204L gene of ASFV gene type I and ASFV gene type II is highly conserved, and 12 specific SNPs sites are identified; the MGF360-9L gene of ASFV gene type I and ASFV gene type II is highly conserved, and 15 specific SNPs sites are identified; the MGF505-11L gene of ASFV gene type I and ASFV gene type II is highly conserved, and 24 specific SNPs sites are identified. The above results show that the B646L gene, CP204L gene, MGF360-9L gene, and MGF505-11L gene can be used as target genes for typing detection of African swine fever virus.

[0085] 2. Screening and identification of SNPs sites of African swine fever virus genotype I, II, and I / II recombinant virus detection target genes

[0086] After identifying the target genes by the above 1, in the total SNPs sites, according to the single nucleotide position and number of SNP sites, further screening and identifying 4 highly specific SNP sites in the highly conserved region of each target gene, a total of 16 SNP sites, for constructing microfluidic sample pretreatment chip specific amplification of African swine fever gene type I, type II, I / II recombinant virus SNPs site. The position and mutation type of 16 SNP sites in the target gene are shown in Table 1 (the position in Table 1 is represented as the name of the target gene: the position on the target gene; the representation of the mutation type is ASFV gene type I / ASFV gene type II or ASFV gene I / II recombinant type). The gene reference version number is NC_044956.1 (Africanswine fever virus Benin 97 / 1 pathogenic isolate, complete genome) and NC_044957.1 (African swine fever virus OURT 88 / 3 avirulent field isolate, complete genome). In the reference target gene, the physical position of the B646L gene is 1941 bp; the length of the CP204L gene is 585 bp; the length of the MGF360-9L gene is 1053 bp; the length of the MGF505-11L gene is 1629 bp.

[0087] Table 1 Single nucleotide polymorphism site information in microfluidic sample pretreatment chip

[0088]

[0089] 3. Establishing LAMP method for specific amplification of SNPs sites of detection target genes of African swine fever gene type I, type II and I / II recombinant virus

[0090] In order to achieve specific amplification of the target gene SNPs site of ASFV gene type I, type II, I / II recombinant detection in the microfluidic sample pretreatment chip, and the sample pretreatment time is significantly shortened from more than 20h to less than 1.5h, avoiding non-specific amplification, simple operation, simple and easy-to-operate instrument equipment, so as to establish LAMP method for each SNP site, the total volume of LAMP reaction system is 20μL, and the components are 0.1μM F3 (forward external primer), 0.1μM B3 (reverse external primer), 0.8μM FIP (forward internal primer), 0.8μM BIP (reverse internal primer), 2U HotStart Bst4.0 DNA polymerase, 20mM MgSO4, 2μL betaine, 3.5mM dNTPs, 75×SYBR Green Dye, ddH2O, 65℃ reaction for 1h, and photographing observation under the full-automatic nucleic acid imaging instrument. The primer sequence of the LAMP method established for each SNP site is shown in Table 2.

[0091] Table 2 LAMP primer sequence for specific amplification of SNPs site

[0092]

[0093]

[0094] 4. Preparation of LAMP system freeze-dried microspheres for specific amplification of target gene SNPs site of African swine fever virus gene type I, type II, I / II recombinant virus detection

[0095] In order to better pre-embed and apply the LAMP system for specific amplification of the target gene SNPs site of ASFV gene type I, type II, I / II recombinant detection in the microfluidic sample pretreatment chip, in order to realize automation, reduce human error, simple operation, and high efficiency and rapidity, the LAMP reaction system is freeze-dried into microspheres. 16 LAMP reaction systems are established for 16 SNP sites of 4 detection target genes. In order to make the amplification and detection results more accurate, the absolute conserved region of ASFV B646L gene is selected as the internal reference, and 1 LAMP reaction system is also established, a total of 17 LAMP reaction systems, and each LAMP reaction system is freeze-dried into microspheres, a total of 17 groups of LAMP reaction system freeze-dried microspheres. The microspheres are white spherical, loose in structure and reticular, can be quickly reconstituted, and stored at room temperature, as shown in Figure 1 .

[0096] 5. Construction of microfluidic sample pretreatment chip

[0097] The above SNP sites and the internal reference can be amplified respectively by PCR tubes, and the genotype of African swine fever virus is typed by genotyping dot chip detection. However, when 16 SNP sites and 1 internal reference are amplified respectively by PCR tubes, there are many operation times, complicated process, complex operation and large human error. Especially in the mixing stage of amplification products, 17 PCR tubes containing a large amount of DNA amplification products need to be opened for each sample detected, which has very serious and unavoidable nucleic acid cross contamination between samples. In order to avoid serious nucleic acid cross contamination between samples, simplify the operation steps, realize one-step sample addition to simultaneously amplify 16 SNP sites and 1 internal reference, and realize automation to reduce human error, a microfluidic sample pretreatment chip (as shown in Figure 2 ) is constructed, wherein A is an elevation view, B is a liquid flow channel layer bottom view, C is a gas flow channel layer top view, and D is a schematic view of a pneumatic valve; 1 is a liquid flow channel layer, 2 is a PDMS elastic film layer, 3 is a gas flow channel layer, 4 is a glass support layer, 5 is a liquid sample addition hole, 6 is a liquid outlet hole, 7 is an air hole, 8 is a peripheral liquid flow channel, 9 is an internal liquid flow channel, 10 is a product collection cavity, 11 is an amplification cavity, 12 is a gas flow channel 1, and 13 is a gas flow channel 2. The specific steps are as follows:

[0098] The main body of the microfluidic sample pretreatment chip is made of PDMS material and is in the shape of a round cake, which is composed of four layers from top to bottom, i.e. a liquid flow channel layer, a PDMS elastic film layer, a gas flow channel layer and a glass support layer (as shown in Figure 2 A);

[0099] The liquid flow channel layer is in the shape of a round cake, and a liquid outlet hole is provided in the middle of the liquid flow channel layer. A peripheral liquid flow channel, 17 amplification cavities, 17 internal liquid flow channels and a product collection cavity are embedded in the top part of the liquid flow channel layer near the PDMS elastic film layer but do not penetrate through. The peripheral liquid flow channel is located at the edge of the bottom of the liquid flow channel layer, and a liquid sample addition hole is provided on the peripheral liquid flow channel, which penetrates through the top of the liquid flow channel layer. The product collection cavity is located around the liquid outlet hole. The 17 amplification cavities are located between the product collection cavity and the peripheral liquid flow channel and are distributed on the same circumference. There are 17 internal liquid flow channels between the product collection cavity and the peripheral liquid flow channel, which penetrate through the 17 amplification cavities and connect the peripheral liquid flow channel and the product collection cavity. The peripheral liquid flow channel and the internal liquid flow channel realize the flow of the sample, so that the sample enters the amplification cavity or collects the sample amplification product. There are two air holes between the peripheral liquid flow channel and the amplification cavity, which penetrate through the liquid flow channel layer and the PDMS elastic film layer and are embedded in but do not penetrate through the bottom of the gas flow channel layer (as shown in Figure 2 B);

[0100] The gas flow channel layer is in the shape of a round cake, and the top is embedded in the bottom of the gas flow channel layer, and gas flow channels 1 and 2 are provided; the position of the gas flow channel 1 corresponds to the middle of the product collection cavity and the amplification cavity of the liquid flow channel layer; the gas flow channel 2 penetrates the gas flow channels 1 and 2; the gas holes, the gas flow channels, and the PDMS elastic film layer constitute the pneumatic valve of the microfluidic sample pretreatment chip; by controlling the gas inflow, the pressure in the gas flow channel 1 is increased, the PDMS elastic film layer is stretched to tightly adhere to the bottom of the internal liquid flow channel, and the annular gas flow channel 1 intersects with the 17 internal liquid flow channels to form 17 pneumatic valves, which realize the closing of the 17 internal liquid flow channels (as shown in Figure 2 Fig. 2C and D).

[0101] In the detection, the LAMP reaction system for amplifying 16 SNP sites and 1 internal reference is freeze-dried and embedded in 17 amplification cavities; the PDMS elastic film layer is stretched to tightly adhere to the bottom of the internal liquid flow channel by controlling the gas nitrogen in the two gas holes, thereby controlling the liquid flow and realizing the closing of the internal liquid flow channel; the amplification of 16 SNP sites and 1 internal reference in the sample DNA and the collection of the amplification products are completed; the liquid outlet is used to collect the amplification products of 16 SNP sites and 1 internal reference in the sample DNA for detection. The pneumatic valve structure is composed of gas holes, gas flow channels, and a PDMS elastic film layer; nitrogen is introduced through the gas flow channel, and under the action of pressure, the stretched PDMS elastic film tightly adheres to the bottom of the liquid flow channel layer, preventing the liquid from flowing (as shown in Figure 2 Fig. 2D).

[0102] Example 2: Application of the microfluidic sample pretreatment chip in specific amplification of African swine fever virus SNP sites

[0103] Based on the highly conserved detection target gene and highly specific SNP site screened in the above-mentioned example 1, the LAMP amplification system for specific amplification of 16 SNP sites of 4 target genes of ASFV is established, and the LAMP reaction system is freeze-dried after microspheres, the LAMP system microspheres are pre-embedded in the microfluidic sample pretreatment chip, the sample is added to the microfluidic sample pretreatment chip for amplification, and when the amplification product presents yellow or red under the full-automatic nucleic acid detector, it indicates that the 16 SNP sites of 4 target genes of ASFV are successfully amplified. The specific steps are as follows:

[0104] (1) Collect clinical samples or field samples, use a virus extraction kit to extract viral DNA (if necessary, first isolate ASFV, and then extract DNA of ASFV), take ddH2O as a blank sample, and extract DNA together with the clinical samples or field samples.

[0105] (2) After connecting the syringe pump with the syringe and the hose, the sample DNA solution is injected into the chip through the liquid sample inlet of the microfluidic sample pretreatment chip. Under the control of the fluid, the sample DNA solution enters the amplification cavity, and the pre-embedded LAMP system microspheres are rapidly reconstituted in the sample DNA solution. Connect the nitrogen cylinder to the air hole, and introduce nitrogen to close the pneumatic valve and prevent liquid flow. The sample DNA amplification reaction is fixed in the amplification cavity. Use the anode oxidation plug that has been pre-removed of nucleic acids to seal each inlet and outlet of the chip. After wrapping the chip with a sealing film, place the chip in a 65℃ water bath and react for 1 hour.

[0106] (3) Open the pneumatic valve, and under the control of the fluid, the amplification solution in the amplification cavity enters the product collection cavity. Use the hose and syringe to collect the amplification product from the liquid outlet. In order to detect the amplification results of each SNP site, only pre-embedded LAMP reaction system microspheres for amplifying one SNP site and a blank control are used in one chip. After all the detection is completed, a new microfluidic sample pretreatment chip is prepared, and LAMP reaction system microspheres for amplifying 16 SNP sites and one internal reference are pre-embedded. The amplification product typing detection is performed. The B646L gene is an internationally recognized gene for typing ASFV gene type I and gene type II, and is also a recombinant gene of ASFV gene type I / II. The recombinant gene belongs to ASFV gene type I, so only the DNA of ASFV gene type I and gene type II is used to amplify and verify the four SNP sites of the B646L gene.

[0107] (4) Take a photo of the amplification product under the automatic nucleic acid detector.

[0108] The detection results are shown in Table 1. Figure 3 As shown in Table 1, the blank control amplification product is colorless or slightly white, and the amplification products of the 16 SNP sites and one internal reference are yellow or red under ultraviolet light, indicating that the 16 SNP sites and one internal reference of the four target genes of ASFV gene type I, II, and I / II recombinant type are successfully amplified by the microfluidic sample pretreatment chip.

[0109] Example 3 Application of microfluidic sample pretreatment chip in sample pretreatment for African swine fever virus detection and typing

[0110] Based on the highly conserved detection target genes and highly specific SNP sites screened in the above embodiment 1, and the microfluidic sample pretreatment chip designed and prepared, the field samples or clinical samples are rapidly treated by one-step sample injection and amplification method through the microfluidic sample pretreatment chip, the amplification products of the 16 SNP sites of the 4 target genes of ASFV are collected on the genotyping dot chip for detection, statistical analysis of the detection results is performed, and the detection samples are typed. When the SNP1-16 sites in the detection results are C, G, C, T, A, C, C, C, C, T, C, C, T, A, A, A in turn, it is ASFV gene type I; when the SNP1-16 sites in the detection results are T, A, T, C, T, T, T, T, A, C, T, T, A, C, C, G in turn, it is ASFV gene type II; when the SNP1-16 sites in the detection results are C, G, C, T, T, T, T, T, A, C, T, T, A, C, C, G in turn, it is ASFV gene I / II recombinant type. The specific steps are as follows:

[0111] (1) Collect clinical samples or field samples, use virus extraction kit to extract viral DNA (if necessary, first isolate ASFV, then extract DNA of ASFV), take ddH2O as a blank sample, and extract DNA together with the clinical samples or field samples.

[0112] (2) Take a microfluidic sample pretreatment chip pre-embedded with LAMP reaction system freeze-dried microspheres amplifying 16 SNP sites and 1 internal reference, inject through the liquid sample hole by using a syringe pump, close the valve, seal the entrances and exits of the chip with an anodized plug, and seal the chip with a sealing film. Then, react in a 65℃ water bath for 1h.

[0113] (3) Collect the amplification products of the 16 SNP sites and 1 internal reference from the outlet hole, detect on the genotyping dot chip, statistically analyze the detection results, and type the detected clinical samples or field samples.

[0114] The detection results are shown in Tables 3, Figure 4 , Figure 5 and Figure 6 , 16 SNP sites and 1 internal reference are successfully clustered and typed, the typing results are consistent with the high-throughput sequencing results, indicating that the microfluidic sample pretreatment chip successfully and specifically amplifies 16 SNP sites and 1 internal reference, and the amplification products are successfully typed. Specifically, Figure 4 , Figure 5 and Figure 6 are the coordinate diagrams of the internal reference and 16 SNP sites of the 4 target genes, and the red or green dots in or near the red or green circles represent the detected SNP sites. By statistically analyzing the bases of these sites in turn, each sample can be successfully typed and identified.

[0115] Table 3 sample typing results

[0116]

[0117] Application of the microfluidic sample pretreatment chip in sample pretreatment for identification of recombinant genes of African swine fever virus genotype I / II recombinant type in Example 4

[0118] Based on the highly conserved detection target genes and highly specific SNP sites screened in Example 1 above, wherein the B646L gene is the currently reported recombinant gene of the ASFV genotype I / II recombinant type, which is derived from the ASFV genotype I, and the CP204L, MGF360-9L, and MGF505-11L genes, after sequence alignment, are derived from the ASFV genotype II in the ASFV genotype I / II recombinant type, therefore, after specific amplification of the 16 SNP sites and 1 internal reference of the sample using the microfluidic sample pretreatment chip, the detection result is that the SNP1-16 sites are C, G, C, T, T, T, T, T, A, C, T, T, A, C, C, G in turn, indicating that the ASFV genotype I / II recombinant type is detected. Among them, when the 4 SNP sites of the B646L gene are C, G, C, T in turn, the 4 SNP sites of the CP204L gene are T, T, T, T in turn, the 4 SNP sites of the MGF360-9L gene are A, C, T, T in turn, and the 4 SNP sites of the MGF505-11L gene are A, C, C, G in turn, it indicates that the recombinant gene of the detected ASFV genotype I / II recombinant type is the B646L gene derived from the ASFV genotype I. And the CP204L, MGF360-9L, and MGF505-11L genes are derived from the ASFV genotype II, that is, the recombinant genes of the ASFV genotype I / II recombinant type are the B646L gene of the ASFV genotype I and the CP204L, MGF360-9L, and MGF505-11L genes of the ASFV genotype II have undergone recombination. The specific steps are as follows:

[0119] (1) Collect clinical samples or field samples, use a virus extraction kit to extract viral DNA (if necessary, first isolate ASFV, then extract DNA of ASFV), take ddH2O as a blank sample, and extract DNA together with the clinical samples or field samples.

[0120] (2) Take a microfluidic sample pretreatment chip pre-embedded with a LAMP reaction system freeze-dried microsphere for amplifying 16 SNP sites and 1 internal reference, inject the sample through the liquid sample hole by using a syringe pump, close the valve, seal the entrances and exits of the chip with an anodized plug, and seal the chip with a sealing film. Then, react in a 65°C water bath for 1 hour.

[0121] (3) Collect the amplification products of 16 SNP sites and 1 internal reference from the liquid outlet hole, detect the 16 SNP sites of the 4 target genes on the genotyping array chip, and identify whether the detection sample is the ASFV gene I / II recombinant type, the recombinant gene, and the recombinant gene is derived from the ASFV gene I type or the ASFV gene II type according to each SNP site.

[0122] The 16 SNP sites of the 4 target genes specifically amplified in the microfluidic sample pretreatment chip prepared by the application have high specificity between types and high conservation within types, so the sample amplified by the microfluidic sample pretreatment chip can also detect that the recombinant gene of the ASFV gene I / II recombinant type is any recombinant between the 4 target genes, for example, the B646L gene and the CP204L gene are derived from the ASFV gene I type, and the MGF360-9L gene and the MGF505-11L gene are derived from the ASFV gene II type.

[0123] Example 5 Application of microfluidic sample pretreatment chip in sample pretreatment for African swine fever virus epidemic and variation monitoring

[0124] Based on the fact that the detection target genes screened in the above-mentioned example 1 are highly conserved in the ASFV strains prevalent since the report of African swine fever, and the SNPs sites are highly conserved within types and highly specific between types, the latest epidemic African swine fever samples are collected in real time, the 16 SNP sites are amplified by the microfluidic sample pretreatment chip, and the detection is performed on the genotyping array chip, and compared with the 4 target genes and 16 SNP sites identified, the epidemic and variation of African swine fever virus in the African swine fever epidemic can be monitored in real time. The specific steps are as follows:

[0125] (1) Collect clinical samples or field samples, use a virus extraction kit to extract viral DNA (if necessary, first isolate ASFV, and then extract DNA of ASFV), take ddH2O as a blank sample, and extract DNA together with the clinical samples or field samples.

[0126] (2) Take a microfluidic sample pretreatment chip pre-embedded with LAMP reaction system freeze-dried microspheres for amplifying 16 SNP sites and 1 internal reference, inject the sample through the liquid sample hole by using a syringe pump, close the valve, seal the entrances and exits of the chip with an anodized plug, and seal the chip with a sealing film. After that, the reaction is carried out in a 65℃ water bath for 1h.

[0127] (3) Collecting the amplification products of 16 SNP sites and 1 internal reference from the liquid outlet hole, detecting 16 SNP sites of 4 target genes on a genotyping array chip, and performing statistical analysis. When the detection results of the 4 SNP sites of the B646L gene are C, G, C, and T, it indicates that the B646L gene does not mutate and belongs to ASFV gene type I. When the detection results of the 4 SNP sites of the B646L gene are T, A, T, and C, it indicates that the B646L gene does not mutate and belongs to ASFV gene type II. When the detection results of the 4 SNP sites of the CP204L gene are A, C, C, and C, it indicates that the CP204L gene does not mutate and belongs to ASFV gene type I. When the detection results of the 4 SNP sites of the CP204L gene are T, T, T, and T, it indicates that the CP204L gene does not mutate and belongs to ASFV gene type II. When the detection results of the 4 SNP sites of the MGF360-9L gene are C, T, C, and C, it indicates that the MGF360-9L gene does not mutate and belongs to ASFV gene type I. When the detection results of the 4 SNP sites of the MGF360-9L gene are A, C, T, and T, it indicates that the MGF360-9L gene does not mutate and belongs to ASFV gene type II. When the detection results of the 4 SNP sites of the MGF505-11L gene are T, A, A, and A, it indicates that the MGF505-11L gene does not mutate and belongs to ASFV gene type I. When the detection results of the 4 SNP sites of the MGF505-11L gene are A, C, C, and G, it indicates that the MGF505-11L gene does not mutate and belongs to ASFV gene type II. Comprehensive analysis of 16 SNP sites of 4 target genes, when the detection results of SNP1-16 are C, G, C, T, A, C, C, C, C, T, C, C, T, A, A, A in turn, it is ASFV gene type I, and no mutation in single nucleotide position and quantity occurs. When the detection results of SNP1-16 are T, A, T, C, T, T, T, T, A, C, T, T, A, C, C, G in turn, it is ASFV gene type II, and no mutation in single nucleotide position and quantity occurs. When the detection results of SNP1-16 are C, G, C, T, T, T, T, T, A, C, T, T, A, C, C, G in turn, it is ASFV gene type I / II recombinant, and the recombinant gene is that the B646L gene of ASFV gene type I and the CP204L, MGF360-9L, and MGF505-11L genes of ASFV gene type II have recombined. The recombinant gene does not mutate, and the single nucleotide position and quantity of the recombinant gene do not mutate.If the 16 SNP sites of the 4 target genes of the I type, II type, I / II recombinant type of ASFV have single nucleotide position and quantity variation, by comparing with the above results, the variation of the ASFV genotype, the specific target gene and the corresponding single nucleotide site can be identified.

Claims

1. The use of a reagent for detecting a SNP site combination in the preparation of a chip / reagent for detecting the genotypes of African swine fever virus type I, type II, and I / II recombinant; the SNP site combination consists of 16 SNP sites, which are respectively: SNP1 : located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 480 of the CDS sequence of the gene, with C / T polymorphism; SNP2: located in the African swine fever virus with the gene version number NC_044956.1 B646L 600th nucleotide of the CDS sequence of the gene, with G / A polymorphism; SNP3: located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 684 of the CDS sequence of the gene, with C / T polymorphism; SNP4: located in the African swine fever virus with the gene version number NC_044956.1 B646L the 711th nucleotide of the gene CDS sequence, with T / C polymorphism; SNP5: located in the African swine fever virus with the gene version number NC_044957.1 CP204L 300th nucleotide of the CDS sequence of the gene, with A / T polymorphism; SNP6: located in the African swine fever virus with the gene version number NC_044957.1 CP204L nucleotide 495 of the CDS sequence of the gene, with the C / T polymorphism; SNP7: located in the African swine fever virus with the gene version number NC_044957.1 CP204L nucleotide 511 of the CDS sequence of the gene, with the C / T polymorphism; SNP8: located in the African swine fever virus with the gene version number NC_044957.1 CP204L the 561st nucleotide of the gene CDS sequence, with C / T polymorphism; SNP9: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L the 659th nucleotide of the CDS sequence of the gene, with C / A polymorphism; SNP10: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L the 717th nucleotide of the CDS sequence of the gene, with T / C polymorphism; SNP11 : located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L nucleotide 752 of the CDS sequence of the gene, with the C / T polymorphism; SNP12: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L 873th nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP13: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L the 45th nucleotide of the CDS sequence of the gene, with T / A polymorphism; SNP14: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 150 of the CDS sequence of the gene, with A / C polymorphism; SNP15: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 770 of the CDS sequence of the gene, with A / C polymorphism; SNP16: Located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L Nucleotide 879 of the CDS sequence of the gene, with A / G polymorphism.

2. The use of a primer for specifically amplifying a SNP site combination in the preparation of a microfluidic sample pretreatment reagent or chip for the genotyping of African swine fever virus type I, type II, and I / II recombinant; the SNP site combination consists of 16 SNP sites, which are respectively: SNP1 : located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 480 of the CDS sequence of the gene, with C / T polymorphism; SNP2: located in the African swine fever virus with the gene version number NC_044956.1 B646L 600th nucleotide of the CDS sequence of the gene, with G / A polymorphism; SNP3: located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 684 of the CDS sequence of the gene, with C / T polymorphism; SNP4: located in the African swine fever virus with the gene version number NC_044956.1 B646L the 711th nucleotide of the CDS sequence of the gene, with T / C polymorphism; SNP5: located in the African swine fever virus with the gene version number NC_044957.1 CP204L 300th nucleotide of the CDS sequence of the gene, with A / T polymorphism; SNP6: located in the African swine fever virus with the gene version number NC_044957.1 CP204L nucleotide 495 of the CDS sequence of the gene, with the C / T polymorphism; SNP7: located in the African swine fever virus with the gene version number NC_044957.1 CP204L the 511th nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP8: located in the African swine fever virus with the gene version number NC_044957.1 CP204L the 561st nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP9: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L the 659th nucleotide of the CDS sequence of the gene, with C / A polymorphism; SNP10: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L the 717th nucleotide of the CDS sequence of the gene, with T / C polymorphism; SNP11 : located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L 752thnucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP12: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L 873th nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP13: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L the 45th nucleotide of the CDS sequence of the gene, with T / A polymorphism; SNP14: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 150 of the CDS sequence of the gene, with A / C polymorphism; SNP15: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 770 of the CDS sequence of the gene, with A / C polymorphism; SNP16: Located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L Nucleotide 879 of the CDS sequence of the gene, with A / G polymorphism.

3. A LAMP reaction system set, characterized by, The LAMP reaction system group includes a primer for specifically amplifying a SNP site combination; the SNP site combination consists of 16 SNP sites, which are respectively: SNP1 : located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 480 of the CDS sequence of the gene, with C / T polymorphism; SNP2: located in the African swine fever virus with the gene version number NC_044956.1 B646L 600th nucleotide of the CDS sequence of the gene, with G / A polymorphism; SNP3: located in the African swine fever virus with the gene version number NC_044956.1 B646L nucleotide 684 of the CDS sequence of the gene, with C / T polymorphism; SNP4: located in the African swine fever virus with the gene version number NC_044956.1 B646L the 711th nucleotide of the gene CDS sequence, with T / C polymorphism; SNP5: located in the African swine fever virus with the gene version number NC_044957.1 CP204L 300th nucleotide of the CDS sequence of the gene, with A / T polymorphism; SNP6: located in the African swine fever virus with the gene version number NC_044957.1 CP204L nucleotide 495 of the CDS sequence of the gene, with the C / T polymorphism; SNP7: located in the African swine fever virus with the gene version number NC_044957.1 CP204L nucleotide 511 of the CDS sequence of the gene, with the C / T polymorphism; SNP8: located in the African swine fever virus with the gene version number NC_044957.1 CP204L the 561st nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP9: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L nucleotide 659 of the CDS sequence of the gene, with C / A polymorphism; SNP10: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L the 717th nucleotide of the CDS sequence of the gene, with T / C polymorphism; SNP11 : located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L 752thnucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP12: located in the African swine fever virus with the gene version number NC_044956.1 MGF360-9L 873th nucleotide of the CDS sequence of the gene, with C / T polymorphism; SNP13: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L the 45th nucleotide of the CDS sequence of the gene, with T / A polymorphism; SNP14: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 150 of the CDS sequence of the gene, with A / C polymorphism; SNP15: located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L nucleotide 770 of the CDS sequence of the gene, with A / C polymorphism; SNP16: Located in the African swine fever virus with the gene version number NC_044956.1 MGF505-11L Nucleotide 879 of the CDS sequence of the gene, with A / G polymorphism.

4. The LAMP reaction system set according to claim 3, wherein The LAMP reaction system group includes LAMP reaction systems 1-16 for specifically amplifying the SNP site combination; The LAMP reaction system 1 includes: a forward external primer shown in SEQ ID NO. 1, a reverse external primer shown in SEQ ID NO. 2, a forward internal primer shown in SEQ ID NO. 3, and a reverse internal primer shown in SEQ ID NO. 4; The LAMP reaction system 2 includes: a forward external primer shown in SEQ ID NO. 5, a reverse external primer shown in SEQ ID NO. 6, a forward internal primer shown in SEQ ID NO. 7, and a reverse internal primer shown in SEQ ID NO. 8; The LAMP reaction system 3 includes: a forward external primer shown in SEQ ID NO. 9, a reverse external primer shown in SEQ ID NO. 10, a forward internal primer shown in SEQ ID NO. 11, and a reverse internal primer shown in SEQ ID NO. 12; The LAMP reaction system 4 comprises: a forward outer primer shown as SEQ ID NO. 13, a reverse outer primer shown as SEQ ID NO. 14, a forward inner primer shown as SEQ ID NO. 15, and a reverse inner primer shown as SEQ ID NO. 16; The LAMP reaction system 5 comprises: a forward outer primer shown as SEQ ID NO. 17, a reverse outer primer shown as SEQ ID NO. 18, a forward inner primer shown as SEQ ID NO. 19, and a reverse inner primer shown as SEQ ID NO. 20; The LAMP reaction system 6 comprises: a forward outer primer shown as SEQ ID NO. 21, a reverse outer primer shown as SEQ ID NO. 22, a forward inner primer shown as SEQ ID NO. 23, and a reverse inner primer shown as SEQ ID NO. 24; The LAMP reaction system 7 comprises: a forward outer primer shown as SEQ ID NO. 25, a reverse outer primer shown as SEQ ID NO. 26, a forward inner primer shown as SEQ ID NO. 27, and a reverse inner primer shown as SEQ ID NO. 28; The LAMP reaction system 8 comprises: a forward outer primer shown as SEQ ID NO. 29, a reverse outer primer shown as SEQ ID NO. 30, a forward inner primer shown as SEQ ID NO. 31, and a reverse inner primer shown as SEQ ID NO. 32; The LAMP reaction system 9 comprises: a forward outer primer shown as SEQ ID NO. 33, a reverse outer primer shown as SEQ ID NO. 34, a forward inner primer shown as SEQ ID NO. 35, and a reverse inner primer shown as SEQ ID NO. 36; The LAMP reaction system 10 comprises: a forward outer primer shown as SEQ ID NO. 37, a reverse outer primer shown as SEQ ID NO. 38, a forward inner primer shown as SEQ ID NO. 39, and a reverse inner primer shown as SEQ ID NO. 40; The LAMP reaction system 11 comprises: a forward outer primer shown as SEQ ID NO. 41, a reverse outer primer shown as SEQ ID NO. 42, a forward inner primer shown as SEQ ID NO. 43, and a reverse inner primer shown as SEQ ID NO. 44; The LAMP reaction system 12 comprises: a forward outer primer shown as SEQ ID NO. 45, a reverse outer primer shown as SEQ ID NO. 46, a forward inner primer shown as SEQ ID NO. 47, and a reverse inner primer shown as SEQ ID NO. 48; The LAMP reaction system 13 comprises a forward external primer shown in SEQ ID NO. 49, a reverse external primer shown in SEQ ID NO. 50, a forward internal primer shown in SEQ ID NO. 51, and a reverse internal primer shown in SEQ ID NO.

52. The LAMP reaction system 14 comprises a forward external primer shown in SEQ ID NO. 53, a reverse external primer shown in SEQ ID NO. 54, a forward internal primer shown in SEQ ID NO. 55, and a reverse internal primer shown in SEQ ID NO.

56. The LAMP reaction system 15 comprises a forward external primer shown in SEQ ID NO. 57, a reverse external primer shown in SEQ ID NO. 58, a forward internal primer shown in SEQ ID NO. 59, and a reverse internal primer shown in SEQ ID NO.

60. The LAMP reaction system 16 comprises a forward external primer shown in SEQ ID NO. 61, a reverse external primer shown in SEQ ID NO. 62, a forward internal primer shown in SEQ ID NO. 63, and a reverse internal primer shown in SEQ ID NO.

64.

5. The LAMP reaction system set according to claim 4, wherein The LAMP reaction system group further comprises a LAMP reaction system 17 for amplifying an internal reference gene, which comprises a forward external primer shown in SEQ ID NO. 65, a reverse external primer shown in SEQ ID NO. 66, a forward internal primer shown in SEQ ID NO. 67, and a reverse internal primer shown in SEQ ID NO.

68.

6. The LAMP reaction system set according to claim 5, wherein The LAMP reaction systems 1-17 are freeze-dried into microspheres.

7. A microfluidic sample preparation chip, characterized in that The microfluidic sample pretreatment chip is embedded with the LAMP reaction system group according to any one of claims 3-6.

8. Use of the LAMP reaction system group according to any one of claims 3-6 or the microfluidic sample pretreatment chip according to claim 7 for non-diagnostic purposes as shown in any one of the following: (1) application in detection and typing of African swine fever virus; (2) application in identification of recombinant genes of African swine fever virus I / II recombinant virus; (3) application in monitoring of African swine fever virus epidemic and variation.

9. A method of identifying non-diagnostic African swine fever virus genotypes, characterized in that, The method comprises: (1) extracting DNA of a sample to be tested; (2) adding the sample DNA into the sample inlet of the microfluidic sample pretreatment chip according to claim 7, and closing the chip and then reacting in a 65℃ water bath for 1 hour; (3) collecting amplification products of 16 SNP sites and 1 internal reference, and detecting on a genotyping array chip; When the sites of SNP1-16 in the detection result are C, G, C, T, A, C, C, C, C, T, C, C, T, A, A, A in turn, the sample is judged as ASFV genotype I; when the sites of SNP1-16 in the detection result are T, A, T, C, T, T, T, T, A, C, T, T, A, C, C, G in turn, the sample is judged as ASFV genotype II; when the sites of SNP1-16 in the detection result are C, G, C, T, T, T, T, T, A, C, T, T, A, C, C, G in turn, the sample is judged as ASFV genotype I / II recombinant.

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