Method for synchronously detecting 11 important epidemic disease pathogens of cattle and sheep

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for synchronously detecting 11 important epidemic disease pathogens of cattle and sheep. Firstly, the invention provides a microfluidic gene chip for detecting 11 important epidemic disease pathogens of cattle and sheep; the gene chip is fixed with detection primers of a goat pox virus, a sheep pox virus, a nodular skin disease virus, an orf virus, a monkey pox virus, an O-type foot and mouth disease virus, an A-type foot and mouth disease virus, an Asia 1-type foot and mouth disease virus, a peste des petits ruminants virus, a New Jersey type vesicular stomatitis virus and an Indiana type vesicular stomatitis virus; secondly, the invention provides a method for simultaneously detecting 11 (serotype / genotype) important epidemic disease pathogens of cattle and sheep, and the method can stably and specifically realize general detection of the 11 (serotype / genotype) important epidemic disease pathogens of cattle and sheep, has the advantages of simplicity, convenience, rapidness, good specificity and high sensitivity, and can be used for high-throughput detection of clinical samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a method for detecting 11 kinds (serotype / genotype) of pathogens, including goat pox virus (GTPV), sheep pox virus (SPPV), lumpy skin disease virus (LSDV), monkeypox virus (MPXV), orofacial ulcer virus (ORFV), foot-and-mouth disease virus type A (FMDV-A), foot-and-mouth disease virus type O (FMDV-O), foot-and-mouth disease virus type Asia 1 (FMDV-Asia1), peste des petits ruminants virus (PPRV), New Jersey vesicular stomatitis virus (VSNJV) and Indiana vesicular stomatitis virus (VSIV). Background Art

[0002] Major animal diseases are still an important factor that seriously restricts the healthy development of the breeding industry. Goat pox, sheep pox and lumpy skin disease are febrile and contact infectious diseases caused by goat pox virus (GTPV), sheep pox virus (SPPV) and lumpy skin disease virus (LSDV) of the genus Capripoxvirus of the subfamily Capripoxvirinae of the family Poxviridae, respectively infecting goats, sheep and cattle. There is a high similarity between GTPV, SPPV and LSDV in terms of pathogenic biology and serological reactions. It is impossible to make differential diagnosis based on pathogen morphology and serological methods, and can only be distinguished by genomic methods. The serotypes that cause foot-and-mouth disease in cattle and sheep in my country include foot-and-mouth disease virus type O (FMDV-O), foot-and-mouth disease virus type A (FMDV-A) and foot-and-mouth disease virus type Asia 1 (FMDV-Asia 1). Among them, foot-and-mouth disease virus type Asia 1 has been effectively controlled and has not been prevalent in the past five years. Vesicular stomatitis (mainly caused by two serotypes, New Jersey vesicular stomatitis virus (VSNJV) and Indiana vesicular stomatitis virus (VSIV)) has not yet been introduced into my country, but it is also an important zoonosis that seriously affects the international cattle industry. Monkeypox is the "most dangerous" infectious disease caused by poxvirus since smallpox was "eradicated". The World Health Organization (WHO) has issued the highest level alert for the second time - a major zoonosis that "constitutes an international public health emergency". Although there is no evidence that monkeypox virus can infect cattle and sheep, as the monkeypox virus mutates, its pathogenicity and infectivity are stronger, and its host range is further expanding, which may become another major animal epidemic affecting the cattle and sheep breeding industry. Sheep ulcer is caused by sheep ulcer virus (ORFV), the most widely distributed zoonosis, and is prevalent in all sheep-raising countries or regions around the world.

[0003] Implementing accurate diagnosis is the primary link in controlling infectious diseases. At present, the detection of these diseases in my country is mainly based on pathogen isolation, serological tests and molecular biological detection methods. Serological methods include neutralization tests and ELISA. These methods can effectively detect these diseases, but cannot detect mixed infections of several pathogens at the same time. Therefore, a method for rapid and efficient detection of these important cattle and sheep diseases is established to provide technical support for the prevention and control of these diseases.

[0004] With the development of molecular biology, the emergence of molecular marker technology has provided a new means for differential diagnosis of viruses with highly homologous genome sequences such as lumpy dermatosis virus, sheeppox virus and goatpox virus. SNP marker is one of the important molecular marker technologies, with the characteristics of large quantity, wide distribution, high stability, easy and fast high-throughput typing. Commonly used SNP detection methods include DNA chip technology, sequencing and competitive allele-specific PCR (KASP). DNA chip technology is the most commonly used method for detecting SNPs, but the cost of this method is relatively high. KASP technology is based on the specific matching of primer terminal bases to type SNPs. This technology has high stability and accuracy. The principle of KASP is to perform accurate double allele judgment on SNPs and InDels at specific sites on a microfluidic chip to achieve SNP / InDel typing. (1) Primer design: design two PCR upstream primers and one universal downstream primer for the two alleles of the target SNP site respectively; (2) SNP identification: template denaturation, upstream PCR primers pair with template DNA, downstream primers pair with the other chain, anneal and extend, and complete the identification of the SNP site; (3) Introduction of label sequence: downstream primers bind and extend to amplify PCR products with label sequences. This step completes the introduction of universal label sequences into the PCR products corresponding to the SNP; (4) Fluorescence signal generation: after PCR amplification, the two universal fluorescent probes containing label sequences in the amplification reagents anneal more to the newly synthesized complementary chains without quenching groups, separate from the quenching groups, emit fluorescence, and generate fluorescent signals. Therefore, the development of microfluidic chip detection methods based on KASP technology can greatly improve the differential diagnosis efficiency of goatpox virus (GTPV), sheeppox virus (SPPV), lumpy skin disease virus (LSDV), orofacial ulcer virus (ORFV), monkeypox virus (MPXV), foot-and-mouth disease virus (FMDV-A, FMDV-O, FMDV-Asia 1), peste des petits ruminants virus (PPRV) and vesicular stomatitis virus (New Jersey type and Indiana type) (VSNJV, VSIV), and improve my country's diagnostic capabilities for major animal epidemics and zoonoses. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a method for detecting 11 kinds (serotypes / genotypes) of important cattle and sheep epidemic pathogens, including goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, type A foot-and-mouth disease virus, type O foot-and-mouth disease virus, Asian type 1 foot-and-mouth disease virus, peste des petits ruminants virus, New Jersey type vesicular stomatitis virus, and Indiana type vesicular stomatitis virus, which specifically includes the following contents:

[0006] In a first aspect, the present invention provides a primer combination for synchronously detecting 11 kinds (serotypes / genotypes) of important cattle and sheep epidemic pathogens, wherein the 11 kinds (serotypes / genotypes) of important cattle and sheep epidemic pathogens include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, Asia 1 foot-and-mouth disease virus, O-type foot-and-mouth disease virus, subtype I foot-and-mouth disease virus, peste des petits ruminants virus, New Jersey vesicular stomatitis virus, and Indiana vesicular stomatitis virus; the primer combination includes primer groups 1-11; the primer group 1 is used for differential diagnosis of sheep pox virus and lumpish skin disease virus, and its nucleotide sequence is shown in SEQ ID NO.1-3; the primer group 2 is used for differential diagnosis of sheep pox virus and goat pox virus, and its nucleotide sequence is shown in SEQ ID NO.4-6; the primer group 3 is used for differential diagnosis of goat pox virus and lumpish skin disease virus, and its nucleotide sequence is shown in SEQ ID NO. NO.7-9; the primer set 4 is used to detect monkeypox virus, and its nucleotide sequence is shown in SEQ ID NO.10-11; the primer set 5 is used to detect sheep sore virus, and its nucleotide sequence is shown in SEQ ID NO.12-13; the primer set 6 is used to detect peste des petits ruminants virus, and its nucleotide sequence is shown in SEQ ID NO.14-15; the primer set 7 is used to detect O type foot-and-mouth disease virus, and its nucleotide sequence is shown in SEQ ID NO.16-17; the primer set 8 is used to detect A type foot-and-mouth disease virus, and its nucleotide sequence is shown in SEQ ID NO.18-19; the primer set 9 is used to detect Asia 1 type foot-and-mouth disease virus, and its nucleotide sequence is shown in SEQ ID NO.20-21; the primer set 10 is used to detect Indiana type vesicular stomatitis virus, and its nucleotide sequence is shown in SEQ ID NO.22-23; the primer set 11 is used to detect New Jersey type vesicular stomatitis virus, and its nucleotide sequence is shown in SEQ ID NO.24-25.

[0007] Preferably, the primer combination also includes an internal control primer group 1 and an internal control primer group 2; the internal control primer group 1 is used to detect the conserved region of the bovine genome sequence, and its nucleotide sequence is shown in SEQ ID NO.26-28; the internal control primer group 2 is used to detect the conserved region of the sheep genome sequence, and its nucleotide sequence is shown in SEQ ID NO.29-31.

[0008] In the second aspect, the present invention provides the use of the primer combination described in the first aspect in the preparation of a product for simultaneously detecting 11 types (serotypes / genotypes) of important cattle and sheep epidemic pathogens, wherein the 11 types (serotypes / genotypes) of important cattle and sheep epidemic pathogens include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, type A foot-and-mouth disease virus, type O foot-and-mouth disease virus, Asian type 1 foot-and-mouth disease virus, peste des petits ruminants virus, New Jersey vesicular stomatitis virus, and Indiana vesicular stomatitis virus.

[0009] In the third aspect, the present invention provides a detection chip, which is obtained by respectively embedding each primer group, positioning point, positive control, and blank control in the primer combination described in the first aspect in different reaction pools of a microfluidic chip; the positioning point is a fluorescent dye that can produce fluorescent signals in both the FAM channel and the HEX channel.

[0010] Preferably, the control substance is a recombinant plasmid of goatpox virus P32 protein gene, sheeppox virus P32 protein gene, lumpy skin disease virus P32 protein gene, sheep sore virus VIR protein gene, monkeypox virus F3L protein gene, peste des petits ruminants virus N protein gene, type A foot-and-mouth disease virus VP1 protein gene, type O foot-and-mouth disease virus VP1 protein gene, Asian type 1 foot-and-mouth disease virus VP1 protein gene, New Jersey vesicular stomatitis virus L protein gene and vesicular stomatitis Indiana virus L protein gene.

[0011] In a fourth aspect, the present invention provides a detection kit, which comprises the primer combination described in the first aspect or the detection chip described in the third aspect.

[0012] Preferably, the kit further comprises a sealing film, a detection probe, a test sample genomic DNA extraction reagent, a PCR amplification reagent, and a KASP reagent.

[0013] In a fifth aspect, the present invention provides a method for detecting 11 kinds (serotypes / genotypes) of important cattle and sheep disease pathogens for the purpose of non-disease diagnosis, wherein the 11 kinds (serotypes / genotypes) of important cattle and sheep disease pathogens include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, type A foot-and-mouth disease virus, type O foot-and-mouth disease virus, Asian type 1 foot-and-mouth disease virus, peste des petits ruminants virus, New Jersey vesicular stomatitis virus, and Indiana vesicular stomatitis virus; the method comprises the following steps:

[0014] (1) Extracting genomic DNA from the sample to be tested;

[0015] (2) respectively mixing the DNA described in step (1) with a PCR amplification reagent, and adding the mixed solution into the injection hole of the detection chip described in the third aspect or the detection chip of the kit described in the fourth aspect;

[0016] (3) sealing the detection chip, centrifuging, heat-sealing, and performing PCR reaction;

[0017] (4) Perform scanning and data analysis.

[0018] Preferably, in step (2), the volume ratio of DNA and reference substance to PCR amplification reagent is 1:1; the amount of the mixed solution added to the injection hole is 38 μL; the PCR reaction program in step (3) is: 95°C for 15 minutes; 95°C for 20 seconds, 61°C-55°C for 60 seconds, 10 cycles, reducing 0.6°C in each cycle; 95°C for 20 seconds, 55°C for 60 seconds, 26 cycles; 37°C for 60 seconds, 1 cycle.

[0019] Preferably, the sample to be tested is selected from any one or more of cattle / sheep blister fluid, eye secretion swabs, oropharyngeal secretion swabs, nasopharyngeal secretion swabs, vaginal secretion swabs, anal secretion swabs, whole blood, plasma, serum, papules / rash, nodules, scabs, lymph nodes, lung / semen, urine, and milk.

[0020] Preferably, the sample to be tested is selected from any one or more of blister fluid, papules (rashes or nodules), and scabs of cattle / sheep.

[0021] The invention has the beneficial effects of: providing a microfluidic gene chip for detecting 11 pathogens of important cattle and sheep diseases, which is fixed with goat pox virus detection primers, sheep pox virus detection primers, nodular skin disease virus detection primers, sheep mouth sore virus detection primers, monkeypox virus detection primers, O-type foot-and-mouth disease virus detection primers, A-type foot-and-mouth disease virus detection primers, Asia 1-type foot-and-mouth disease virus detection primers, peste des petits ruminants virus detection primers, New Jersey-type vesicular stomatitis virus detection primers and Indiana-type vesicular stomatitis virus detection primers; and the invention also provides a method for detecting 11 pathogens of important cattle and sheep diseases. The invention can stably and specifically realize the universal detection of 11 (serotype / genotype) disease viruses, is simple, fast, has good specificity and high sensitivity, and can be used for the detection of clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram of a specific array of a microfluidic chip for embedding primers;

[0023] Figure 2 The microfluidic chip scanning results of samples 1 to 16 in Example 3. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] The primer sequences in the examples are all in the 5′-3′ direction.

[0027] Example 1 Primer combinations for detecting 11 important pathogens of cattle and sheep diseases

[0028] The present invention uses goatpox virus P32 protein gene, sheeppox virus P32 protein gene, nodular skin disease virus P32 protein gene, sheep sore virus VIR protein gene, monkeypox virus F3L protein gene, peste des petits ruminants virus N protein gene, A-type foot-and-mouth disease virus VP1 protein gene, O-type foot-and-mouth disease virus VP1 protein gene, Asian type 1 foot-and-mouth disease virus VP1 protein gene, New Jersey type vesicular stomatitis virus L protein gene and vesicular stomatitis Indiana type virus L protein gene as target genes, and designs primer sets according to the competitive allele-specific PCR principle, specifically:

[0029] Primer set 1 is used for differential diagnosis of sheep pox virus and bovine lumpy skin disease virus, and is composed of three primers based on the principle of competitive allele-specific PCR, namely, upstream primer 1 (SEQ ID NO.1), upstream primer 2 (SEQ ID NO.2) and downstream primer (SEQ ID NO.3) specific for the P32 protein gene of sheep pox virus and lumpy skin disease virus, and is used to distinguish the A / C at the SNP position 670bp of the P32 protein gene of the two;

[0030] Primer set 2 is used for differential diagnosis of sheeppox virus and goatpox virus, and is composed of three primers based on the principle of competitive allele-specific PCR, namely, upstream primer 1 (SEQ ID NO.4), upstream primer 2 (SEQ ID NO.5) and downstream primer (SEQ ID NO.6) specific for the P32 protein gene of sheeppox virus and goatpox virus, which are used to distinguish the A / C at the SNP position 670bp of the P32 protein gene of the two;

[0031] Primer set 3 is used for differential diagnosis of goat pox virus and lumpy skin disease virus, and is composed of three primers based on the principle of competitive allele-specific PCR, namely, upstream primer 1 (SEQ ID NO.7), upstream primer 2 (SEQ ID NO.8) and downstream primer (SEQ ID NO.9) specific for the P32 protein gene of goat pox virus and lumpy skin disease virus, which are used to distinguish the T / C at the SNP position 551bp of the P32 protein gene of the two;

[0032] Primer set 4 is used to detect monkeypox virus, and is composed of two primers based on the principle of competitive allele-specific PCR, namely, upstream primer 1 (SEQ ID NO.10) and downstream primer (SEQ ID NO.11) specific for the monkeypox virus F3L protein gene, used to detect T at the SNP position 350 bp of the F3L protein gene;

[0033] Primer set 5 is used to detect the stomatitis virus, and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.12) and a downstream primer (SEQ ID NO.13) specific for the stomatitis virus VIR protein gene, which are used to detect the G at the SNP position 397 bp of the VIR protein gene;

[0034] Primer set 6 is used to detect peste des petits ruminants virus, and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.14) and a downstream primer (SEQ ID NO.15) specific for the N protein gene of peste des petits ruminants virus, and is used to detect A at the SNP position 822 bp of the N protein gene;

[0035] Primer set 7 is used to detect O-type foot-and-mouth disease virus, and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.16) and a downstream primer (SEQ ID NO.17) specific for the VP1 protein gene of the foot-and-mouth disease virus, and is used to detect C at the SNP position 2476 bp of the O-type foot-and-mouth disease virus genome;

[0036] Primer set 8 is used to detect type A foot-and-mouth disease virus (FMDV-A), and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.18) and a downstream primer (SEQ ID NO.19) specific for the VP1 protein gene of the foot-and-mouth disease virus, and is used to detect the G at the SNP position 3781 bp of the type A foot-and-mouth disease virus genome;

[0037] Primer set 9 is used to detect Asia 1 foot-and-mouth disease virus, and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.20) and a downstream primer (SEQ ID NO.21) specific for the VP1 protein gene of foot-and-mouth disease virus, and is used to detect C at the SNP position 3085bp of the Asia 1 foot-and-mouth disease virus genome;

[0038] Primer set 10 is used to detect Indiana vesicular stomatitis virus (VSIV), and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.22) and a downstream primer (SEQ ID NO.23) specific for the L protein gene of vesicular stomatitis virus, and is used to detect A at the SNP position 3239 bp of the L protein gene;

[0039] Primer set 11 is used to detect New Jersey vesicular stomatitis virus (VSNJV), and is composed of two primers based on the principle of competitive allele-specific PCR, namely, an upstream primer (SEQ ID NO.24) and a downstream primer (SEQ ID NO.25) specific for the L protein gene of vesicular stomatitis virus, and is used to detect A at the 400bp SNP position of the L protein gene;

[0040] The internal control primer set is used to detect the conserved region of the cattle and sheep genome sequence. In the process of testing real samples, positive or negative test results appear.

[0041] Internal control primer set 1 is used to detect the conserved region of the bovine genome sequence, and is composed of three primers based on the principle of competitive allele-specific PCR, namely, bovine housekeeping gene-specific upstream primer 1 (SEQ ID NO.26), upstream primer 2 (SEQ ID NO.27) and downstream primer (SEQ ID NO.28), which are used to detect A at the SNP position 7766bp of the bovine genome sequence;

[0042] Internal control primer set 2 is used to detect the conserved region of the sheep genome sequence. It consists of three primers based on the principle of competitive allele-specific PCR, namely, sheep housekeeping gene-specific upstream primer 1 (SEQ ID NO.29), upstream primer 2 (SEQ ID NO.30) and downstream primer (SEQ ID NO.31), which are used to detect C at the SNP position 8469bp of the sheep genome sequence.

[0043] The above primers are used to detect 11 (serotypes / genotypes) of important cattle and sheep epidemic pathogens (all consisting of 1 or 2 upstream primers and 1 downstream primer).

[0044] Example 2 Gene chip and detection kit

[0045] Based on the primer combination described in Example 1, the present invention has developed a gene chip and a detection kit for detecting the above 11 pathogens.

[0046] 1. Preparation of Microfluidic Chip with Primers

[0047] The microfluidic chip with embedded primers has 112 reaction pools (array format is 4 columns × 28 rows). The target is embedded in the reaction pool of the microfluidic chip by using agarose aqueous solution as an embedding agent (it must be dried after embedding). The microfluidic chip with embedded primers has 4 detection channels, each column is a detection channel, each detection channel has 1 injection hole and 1 output hole, and each detection channel can complete the detection of one sample. The targets embedded in the 112 reaction pools are as follows:

[0048] The four reaction pools in the first row are all embedded with positioning points; the positioning points are fluorescent dyes that can produce fluorescent signals in both the FAM channel and the HEX channel;

[0049] The four reaction pools in the second row are all embedded with a blank control primer set; the blank control primer set is a blank control without primers;

[0050] The four reaction pools in the third row are all embedded with the internal control primer set 1 described in Example 1;

[0051] The 4 reaction pools in the 4th row are all embedded with the internal control primer set 2 described in Example 1;

[0052] The four reaction pools in the fifth row all contain the primer set 1 described in Example 1;

[0053] The four reaction pools in the sixth row all contain the primer set 1 described in Example 1;

[0054] The four reaction pools in row 7 all contain primer set 2 described in Example 1;

[0055] The four reaction pools in row 8 are all embedded with primer set 2 described in Example 1;

[0056] The four reaction pools in row 9 all contain primer set 3 described in Example 1;

[0057] The four reaction pools in row 10 all contain primer set 3 described in Example 1;

[0058] The four reaction pools in row 11 are all embedded with primer set 4 described in Example 1;

[0059] The four reaction pools in row 12 are all embedded with primer set 4 described in Example 1;

[0060] The four reaction pools in row 13 all contain primer set 5 described in Example 1;

[0061] The four reaction pools in row 14 all contain primer set 5 described in Example 1;

[0062] The four reaction pools in row 15 are all embedded with primer set 6 described in Example 1;

[0063] The four reaction pools in row 16 are all embedded with primer set 6 described in Example 1;

[0064] The four reaction pools in row 17 all contain primer set 7 described in Example 1;

[0065] The four reaction pools in row 18 all contain primer set 7 described in Example 1;

[0066] The four reaction pools in row 19 all contain primer set 8 described in Example 1;

[0067] The four reaction pools in row 20 all contain primer set 8 described in Example 1;

[0068] The four reaction pools in row 21 all contain primer set 9 described in Example 1;

[0069] The four reaction pools in row 22 all contain primer set 9 described in Example 1;

[0070] The four reaction pools in row 23 are all embedded with the primer set 10 described in Example 1;

[0071] The four reaction pools in row 24 are all embedded with the primer set 10 described in Example 1;

[0072] The four reaction pools in row 25 are all embedded with the primer set 11 described in Example 1;

[0073] The four reaction pools in row 26 are all embedded with the primer set 11 described in Example 1;

[0074] The 4 reaction pools in the 27th row are all embedded with the detection index primers and templates of this kit, i.e., the primer sets 1-11 for the 11 important cattle and sheep disease pathogens described in Example 1 and the recombinant plasmids expressing goatpox virus P32 protein gene, sheeppox virus P32 protein gene, nodular skin disease virus P32 protein gene, sheep sore virus VIR protein gene, monkeypox virus F3L protein gene, peste des petits ruminants virus N protein gene, type A foot-and-mouth disease virus VP1 protein gene, type O foot-and-mouth disease virus VP1 protein gene, Asian type 1 foot-and-mouth disease virus VP1 protein gene, New Jersey vesicular stomatitis virus L protein gene and vesicular stomatitis Indiana virus L protein gene as positive controls, used for chip positive control;

[0075] The four reaction pools in the 28th row are all embedded with positioning points, which are the same as those in the first row and are fluorescent dyes that can produce fluorescent signals in both the FAM channel and the HEX channel.

[0076] The embedding amount of each primer of each primer set in the corresponding single reaction pool is as follows: 12 μM upstream primer, 30 μM downstream primer. Figure 1 .

[0077] The above primer combination is used to amplify the probe tag sequence connected to the primer by recognizing the sequence in the template DNA. The KASP reagent contains two universal probes, which correspond to the tag sequences in the primer set. When the two universal probes in the KASP reagent recognize the tag sequence on the primer, the quenching group is turned on to generate a fluorescent signal. The amplified chip is scanned by a dedicated chip scanner to scan the fluorescence in each reaction pool. If the template DNA contains specific genes and sites corresponding to the pathogen, FAM fluorescence (red) or HEX fluorescence (bright green) will be displayed.

[0078] 2. Preparation of the kit

[0079] The kit includes the following components: a microfluidic chip with embedded primers prepared in step 1, a PCR amplification reagent, a sealing film and a control substance.

[0080] PCR amplification reagent: KASP TF V4.0 2×Master mix (LGC, catalog number KBS-1016-002).

[0081] The control substance is a recombinant plasmid constructed and sequenced to express the goatpox virus P32 protein gene, sheeppox virus P32 protein gene, lumpy skin disease virus P32 protein gene, sheep sore virus VIR protein gene, monkeypox virus F3L protein gene, peste des petits ruminants virus N protein gene, type A foot-and-mouth disease virus VP1 protein gene, type O foot-and-mouth disease virus VP1 protein gene, type 1 Asian foot-and-mouth disease virus VP1 protein gene, New Jersey vesicular stomatitis virus L protein gene, and vesicular stomatitis Indiana virus L protein gene.

[0082] 3. Experimental methods of the kit

[0083] (1) Extracting genomic DNA from the sample to be tested (blood, blister fluid, swab, etc.);

[0084] (2) Mix 20 μL of genomic DNA (DNA content 60-400 ng) and 20 μL of PCR amplification reagent, and then add 38 μL of the mixture to the injection hole of the microfluidic chip with embedded primers. This is the operation for a single injection hole, and multiple injection holes can be operated in parallel.

[0085] (3) After completing step (2), seal with sealing film, centrifuge at 4000 rpm for 60 seconds, and then perform PCR reaction. PCR reaction program: 95°C for 15 minutes; 95°C for 20 seconds, 61°C-55°C (0.6°C reduction per cycle) for 60 seconds, 10 cycles; 95°C for 20 seconds, 55°C for 60 seconds, 26 cycles; 37°C for 60 seconds, 1 cycle.

[0086] (4) After completing step 3, the chip is scanned and the data is analyzed. The chip interpretation principles and standards are shown in Table 1.

[0087] Table 1 Chip interpretation principles

[0088]

[0089] Example 3 Detection using a kit

[0090] The kit prepared in Example 2 was verified using samples or strains with known backgrounds (see Table 2).

[0091] Table 2 Test sample information and chip test results

[0092]

[0093] Mini BEST Viral RNA / DNA Extraction Kit (TaKaRa, Cat. No. 9766) was used to extract total viral RNA / DNA from 16 clinical samples of cattle and sheep that had been confirmed by PCR and sequencing, and then PrimeScript TM IV 1st strand cDNA Synthesis Mix was used to reverse transcribe and synthesize cDNA, which were named sample 1 to sample 16 in sequence.

[0094] Samples 1 to 16 and the control substance in the kit (with a concentration of 3 ng / μl) were used as templates and the kit was used for detection (the method is shown in Example 2).

[0095] Results Figure 2 The results showed that the method and kit of the present invention can be used for goat pox virus, sheep pox virus, lumpy skin disease virus, sheep sore virus, O-type foot-and-mouth disease virus and Asia type 1 foot-and-mouth disease virus, and can meet the needs of diagnosing important epidemic diseases of cattle and sheep.

[0096] Example 4 Detection of clinical samples

[0097] The kit prepared in Example 2 was used and operated according to the instructions for use to test 55 clinical samples. Among the 55 clinical samples, 4 were collected from whole blood samples of healthy cattle and sheep, and genomic RNA and DNA were extracted respectively, which were named cattle DNA, cattle cDNA, sheep DNA and sheep cDNA respectively; the remaining 51 clinical samples were all tested by PCR and sequencing analysis to confirm the pathogens contained. The clinical sample information is shown in Table 3. The results show that the test results of the 51 clinical samples were 100% consistent with the PCR test and sequencing test results.

[0098] Table 3 Clinical sample information and chip test results

[0099]

[0100]

[0101] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer combination for synchronously detecting 11 pathogens of important cattle and sheep diseases, wherein the 11 pathogens of important cattle and sheep diseases include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, foot-and-mouth disease virus type A, foot-and-mouth disease virus type O, foot-and-mouth disease virus type Asia 1, peste des petits ruminants virus, New Jersey vesicular stomatitis virus, and Indiana vesicular stomatitis virus; characterized in that, The primer combination includes primer sets 1-11; The primer set 1 is used for differential diagnosis of sheeppox virus and lumpy skin disease virus, and its nucleotide sequence is shown in SEQ ID NO.1-3; the primer set 2 is used for differential diagnosis of sheeppox virus and goatpox virus, and its nucleotide sequence is shown in SEQ ID NO.4-6; the primer set 3 is used for differential diagnosis of goatpox virus and lumpy skin disease virus, and its nucleotide sequence is shown in SEQ ID NO.7-9; the primer set 4 is used for detecting monkeypox virus, and its nucleotide sequence is shown in SEQ ID NO.10-11; the primer set 5 is used for detecting sheep sore virus, and its nucleotide sequence is shown in SEQ ID NO.12-13; the primer set 6 is used for detecting peste des petits ruminants virus, and its nucleotide sequence is shown in SEQ ID NO.14-15; the primer set 7 is used for detecting O-type foot-and-mouth disease virus, and its nucleotide sequence is shown in SEQ ID NO.16-17; the primer set 8 is used for detecting A-type foot-and-mouth disease virus, and its nucleotide sequence is shown in SEQ ID NO.18-19; the primer set 9 is used for detecting Asia Type 1 foot-and-mouth disease virus, whose nucleotide sequence is shown in SEQ ID NO.20-21; the primer set 10 is used to detect Indiana type vesicular stomatitis virus, whose nucleotide sequence is shown in SEQ ID NO.22-23; the primer set 11 is used to detect New Jersey type vesicular stomatitis virus, whose nucleotide sequence is shown in SEQ ID NO.24-25.

2. The primer combination according to claim 1, characterized in that The primer combination also includes an internal control primer group 1 and an internal control primer group 2; the internal control primer group 1 is used to detect the conserved region of the bovine genome sequence, and its nucleotide sequence is shown in SEQID NO.26-28; the internal control primer group 2 is used to detect the conserved region of the sheep genome sequence, and its nucleotide sequence is shown in SEQID NO.29-31.

3. Use of the primer combination as described in claim 1 or 2 in preparing a product for synchronously detecting 11 kinds of important cattle and sheep disease pathogens, wherein the 11 kinds of important cattle and sheep disease pathogens include goat pox virus, sheep pox virus, lumpy skin disease virus, monkeypox virus, sheep sore virus, type A foot-and-mouth disease virus, type O foot-and-mouth disease virus, Asia 1 type foot-and-mouth disease virus, peste des petits ruminants virus, New Jersey type vesicular stomatitis virus, and Indiana type vesicular stomatitis virus.

4. A detection chip, characterized in that: The detection chip is obtained by respectively embedding each primer group in the primer combination described in claim 1 or 2, as well as the positioning point, the positive control, and the blank control in different reaction pools of a microfluidic chip; the positioning point is a fluorescent dye that can produce fluorescent signals in both the FAM channel and the HEX channel.

5. The chip according to claim 4, characterized in that: The positive control is a recombinant plasmid of goatpox virus P32 protein gene, sheeppox virus P32 protein gene, nodular skin disease virus P32 protein gene, sheep oropharyngeal canker virus VIR protein gene, monkeypox virus F3L protein gene, peste des petits ruminants virus N protein gene, type A foot-and-mouth disease virus VP1 protein gene, type O foot-and-mouth disease virus VP1 protein gene, Asian type 1 foot-and-mouth disease virus VP1 protein gene, New Jersey vesicular stomatitis virus L protein gene and vesicular stomatitis Indiana virus L protein gene, which is verified by sequencing and constructed in the pMD19-T cloning vector.

6. A detection kit, characterized in that: The kit comprises the primer combination described in any one of claims 1-2 or the detection chip described in any one of claims 4-5.

7. The kit according to claim 6, characterized in that The kit also includes a sealing film, a detection probe, a test sample genome DNA extraction reagent, a PCR amplification reagent, and a KASP reagent.

8. A method for detecting 11 pathogens of important cattle and sheep diseases for the purpose of non-disease diagnosis, wherein the 11 pathogens of important cattle and sheep diseases include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, sheep sore virus, foot-and-mouth disease virus type A, foot-and-mouth disease virus type O, foot-and-mouth disease virus type Asia 1, peste des petits ruminants virus, New Jersey vesicular stomatitis virus, and Indiana vesicular stomatitis virus; characterized in that: The method comprises the following steps: (1) Extracting the genome of the sample to be tested; (2) respectively mixing the DNA obtained in step (1) with a PCR amplification reagent, and adding the mixed solution into the injection hole of the detection chip of claim 4 or the detection chip of the kit of any one of claims 5 to 7; (3) sealing the detection chip, centrifuging, heat-sealing, and performing PCR reaction; (4) Perform scanning and data analysis.

9. The method according to claim 8, characterized in that In step (2), the volume ratio of DNA and reference substance to PCR amplification reagent is 1:1; the amount of the mixed solution added to the injection hole is 38 μL; the PCR reaction program in step (3) is: 95°C for 15 minutes; 95°C for 20 seconds; 61°C-55°C for 60 seconds, 10 cycles, reducing 0.6°C in each cycle; 95°C for 20 seconds, 55°C for 60 seconds, 26 cycles; 37°C for 60 seconds, 1 cycle.

10. The method according to claim 8 or 9, characterized in that The sample to be tested is selected from any one or more of cattle / sheep blister fluid, eye secretion swabs, oropharyngeal secretion swabs, nasopharyngeal secretion swabs, vaginal secretion swabs, anal secretion swabs, whole blood, plasma, serum, papules / rashes, nodules, scabs, lymph nodes, lung / semen, urine, and milk.

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