Method for synchronously detecting six poxviruses

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for synchronously detecting six poxviruses. Firstly, the invention provides a microfluidic gene chip for synchronously detecting six pox viruses, and the gene chip is fixed with detection primers for goat pox virus, sheep pox virus, nodular skin disease virus, orf virus, monkey pox virus and vaccinia virus; secondly, the invention provides a method for simultaneously detecting six different poxviruses, and the method can stably and specifically realize general detection of six poxviruses infecting human, 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 synchronously detecting six poxviruses, namely goat poxvirus (GTPV), sheep poxvirus (SPPV), lumpy skin disease virus (LSDV), monkeypox virus (MPXV), cowpox virus (CPXV) and oropharyngeal canker virus (ORFV). Background Art

[0002] Poxviridae is a dsDNA virus with the largest genome, the most complex structure, the widest host range, and the most species that infects humans and animals. It is divided into the subfamily of Chordatapoxvirinae and the subfamily of Entomopoxvirinae. The subfamily of Chordata includes 18 confirmed virus genera, such as Orthopoxvirus, Parapoxvirus, and Capripoxvirus. To date, a total of 47 poxviruses have been discovered and confirmed, and most of the members are pathogens that seriously endanger the health of humans and animals. Among them, Monkeypox virus (MPXV) is the "most dangerous" poxvirus after the global eradication of smallpox, and Orfvirus (ORFV) is the most widespread zoonotic poxvirus. Both Monkeypox virus and Orfvirus are listed by the US CDC as poxviruses that pose a serious threat to human health. Lumpy skin disease, sheeppox and goatpox are caused by lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox virus (GTPV), respectively, and are the most serious infectious diseases among all animal poxvirus diseases.

[0003] Monkeypox virus (MPXV) is the pathogen that causes monkeypox (mpox). It belongs to the same genus as smallpox virus, and can cause clinical symptoms similar to smallpox, including fever and rash, infecting humans. Although the pathogenicity of monkeypox virus is far less than that of smallpox virus, its host range is very wide: almost all rodents such as marmots, West African squirrels, Cuvier's African squirrels, red-legged sun squirrels, striped squirrels, hazel dormice, anteaters, rats, mice and rabbits are its reservoir hosts; mammals such as African elephants, wild boars, porcupines and antelopes, as well as all non-human primates are its natural hosts; humans can be infected after contact with infected animals, and it has the characteristics of human-to-human transmission. Compared with the characteristic of smallpox virus that only infects humans, monkeypox is more difficult to eliminate and eradicate.

[0004] Oral ulcer virus (ORFV) is a member of the genus Parapoxvirus and is the pathogen that causes contagious impetigo (also known as oral ulcer) in goats and sheep. Oral ulcer is an acute, highly contact, epitheliotropic zoonotic infectious disease. Oral ulcer virus mainly infects goats and sheep, and can also infect reindeer, squirrels, musk oxen, Japanese serow and many other wild animals. Breeders and slaughterhouse workers are infected with oral ulcer virus when their skin and mucous membranes are damaged and they have close contact with sick animals. Oral ulcer is prevalent in various sheep-raising countries or regions such as the Americas, Europe, Africa and Asia. The disease is currently the most widespread zoonotic poxvirus disease, which seriously threatens the development of the sheep industry in various countries and human health.

[0005] Lumpy skin disease virus, sheeppox virus and goatpox virus are all members of the genus Goatpoxvirus. The three have extensive serological cross-reaction and cross-protection. The whole genome nucleotide sequences are highly similar, with a homology of more than 94.9%; they cannot be distinguished morphologically and serologically, and can only be distinguished through genome sequencing; they have relatively strict species specificity, that is, in the natural state, lumpy skin disease virus only infects cattle, sheeppox virus only infects sheep, and goatpox virus only infects goats. Among them, sheeppox and goatpox are animal diseases that must be reported to the World Organization for Animal Health (OIE), listed as one of the ten major animal diseases causing poverty by the Food and Agriculture Organization of the United Nations (FAO), and are now listed as Class II animal infectious diseases in my country. Lumpy skin disease is a febrile, systemic, contact-borne insect-borne infectious disease. Blood-sucking arthropods such as mosquitoes, flies, midges and ticks play a key role in the spread of lumpy skin disease virus. In the past 10 years, the prevalence and harm of nodular dermatosis in Eurasia have become increasingly serious. It is now found in almost all countries or regions in Africa, Asia, and southeastern Europe. It is one of the animal diseases that must be reported to the OIE, and my country has designated it as a Class II animal disease.

[0006] Implementing accurate diagnosis is the first step 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, it is very necessary to establish a method for rapid and efficient detection of these 6 different poxviruses for the prevention and control of these diseases.

[0007] 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), monkeypox virus (MPXV), cowpox virus (CPXV) and oropharyngeal canker virus (ORFV), and improve my country's diagnostic technology capabilities for major animal epidemics and zoonoses. Summary of the invention

[0008] In view of the above technical problems, the object of the present invention is to provide a method for detecting six different poxviruses including goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep sore virus, which specifically includes the following contents:

[0009] In the first aspect, the present invention provides a primer combination for synchronously detecting 6 different poxviruses, wherein the 6 poxviruses include goatpox virus, sheeppox virus, lumpy skin disease virus, monkeypox virus, cowpox virus and sheep sore virus; the primer combination includes primer groups 1-5; the primer group 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 group 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 group 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 group 4 is used for universal detection of monkeypox virus and cowpox virus, and its nucleotide sequence is shown in SEQ ID NO.10-11; the primer group 5 is used for detecting sheep sore virus, and its nucleotide sequence is shown in SEQ ID NO.12-13.

[0010] Preferably, the primer combination also includes internal control primer group 1, internal control primer group 2 and internal control primer group 3; 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.14-16; 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.17-19; the internal control primer group 3 is used to detect the conserved region of the human genome sequence, and its nucleotide sequence is shown in SEQ ID NO.20-22.

[0011] In a 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 six different poxviruses, wherein the six poxviruses include goatpox virus, sheeppox virus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep pox virus.

[0012] In the third aspect, the present invention provides a detection chip, which is obtained by respectively embedding each primer group in the primer combination described in the first aspect, as well as the positioning point, positive control, and 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.

[0013] Preferably, the positive control substance is a recombinant plasmid of goatpox virus P32 gene, sheeppox virus P32 gene, lumpy skin disease virus P32 gene, monkeypox virus and cowpox virus F3L gene, and sheep pox virus VIR gene fragment.

[0014] 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.

[0015] 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.

[0016] In a fifth aspect, the present invention provides a method for detecting six different poxviruses for non-disease diagnosis purposes, wherein the six poxviruses include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep sore virus; the method comprises the following steps:

[0017] (1) Extracting genomic DNA from the sample to be tested;

[0018] (2) respectively mixing the genomic 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;

[0019] (3) sealing the detection chip, centrifuging, heat-sealing, and performing PCR reaction;

[0020] (4) Perform scanning and data analysis.

[0021] Preferably, in step (2), the volume ratio of genomic 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.

[0022] 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; and any one or more of human blister fluid, eye secretion swabs, oropharyngeal secretion swabs, nasopharyngeal secretion swabs, whole blood, plasma, serum, papules / rash, nodules, and scabs.

[0023] Preferably, the sample to be tested is selected from any one or more of blister fluid, papules (rashes or nodules), and scabs of humans, cattle, and sheep.

[0024] The beneficial effects of the present invention are as follows: the present invention provides a microfluidic gene chip for detecting 6 different poxviruses, which is fixed with primers for detecting goat poxvirus, sheep poxvirus, nodular skin disease virus, monkeypoxvirus, cowpoxvirus and sheep sore virus; and the present invention also provides a method for detecting 6 different poxviruses. The universal detection of 6 poxviruses can be stably and specifically realized, and the method is simple, fast, has good specificity and high sensitivity, and can be used for the detection of clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of a specific array of a microfluidic chip for embedding primers;

[0026] Figure 2 These are the microfluidic chip scanning results of samples 1 to 15 in Example 3. DETAILED DESCRIPTION

[0027] 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.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] The primer sequences in the examples are all in the 5'-3' direction.

[0030] Example 1 Primer combinations for detecting 6 poxviruses

[0031] The present invention uses goatpox virus P32 gene, sheeppox virus P32 gene, nodular skin disease virus P32 gene, sheep sore virus VIR gene, monkeypox virus and cowpox virus F3L gene as target genes, and designs primer sets according to the principle of competitive allele-specific PCR, specifically:

[0032] 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, sheep pox virus and lumpy skin disease virus P32 gene specific upstream primer 1 (SEQ ID NO.1), upstream primer 2 (SEQ ID NO.2) and downstream primer (SEQ ID NO.3), which are used to distinguish the A / C of the 670bp SNP position of the P32 gene of the two;

[0033] 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 gene of sheeppox virus and goatpox virus, which are used to distinguish the A / C at the 670bp SNP position of the P32 gene of the two;

[0034] 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 gene of goat pox virus and lumpy skin disease virus, which are used to distinguish the T / C at the 551 bp of the P32 gene SNP position of the two;

[0035] Primer set 4 is used for universal detection of monkeypox virus and cowpox virus, and consists 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 gene, used to detect T at the 350 bp position of the SNP of the F3L gene;

[0036] 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 gene, and is used to detect the G at the 397 bp SNP position of the VIR gene;

[0037] The internal control primer set is used to detect the conserved regions of human, cattle and sheep genome sequences, and positive or negative test results appear during the detection of real samples.

[0038] 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.14), upstream primer 2 (SEQ ID NO.15) and downstream primer (SEQ ID NO.16), which are used to detect A at the SNP position 7766bp of the bovine genome sequence;

[0039] 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.17), upstream primer 2 (SEQ ID NO.18) and downstream primer (SEQ ID NO.19), which are used to detect C at the SNP position 8469bp of the sheep genome sequence.

[0040] Internal control primer set 3 is used to detect the conserved region of the human genome sequence. It consists of three primers based on the principle of competitive allele-specific PCR, namely, human housekeeping gene-specific upstream primer 1 (SEQ ID NO.20), upstream primer 2 (SEQ ID NO.21) and downstream primer (SEQ ID NO.22), which are used to detect G at the SNP position 892bp of the human gene sequence.

[0041] The above primers are used to detect 6 types of poxviruses (each consisting of 1 or 2 upstream primers and 1 downstream primer).

[0042] Example 2 Gene chip and detection kit

[0043] 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 6 poxviruses.

[0044] 1. Preparation of Microfluidic Chip with Primers

[0045] 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:

[0046] 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;

[0047] The 4 reaction pools in the 2nd and 3rd rows are all embedded with blank control primer sets; the blank control primer set is a blank control without primers;

[0048] The 4 reaction pools in the 4th and 5th rows are all embedded with the internal control primer set 1 described in Example 1;

[0049] The four reaction pools in the 6th and 7th rows all contain the internal control primer set 2 described in Example 1;

[0050] The four reaction pools in rows 8 and 9 all contain the internal control primer set 3 described in Example 1;

[0051] The four reaction pools in rows 10-12 all contain the primer set 1 described in Example 1;

[0052] The four reaction pools in rows 13 to 15 are all embedded with the primer set 2 described in Example 1;

[0053] The four reaction pools in rows 16 to 18 all contain primer set 3 described in Example 1;

[0054] The four reaction pools in rows 19 to 21 all contain primer set 4 described in Example 1;

[0055] The four reaction pools in rows 22 to 24 all contain primer set 5 described in Example 1;

[0056] The 4 reaction pools in row 25 are all embedded with a blank control primer set, which is a blank control without primers;

[0057] The four reaction pools in the 26th and 27th rows are all embedded with the detection index primers and templates of the kit, i.e., the six poxvirus primer sets 1-5 described in Example 1 and the recombinant plasmids expressing the goat poxvirus P32 gene, sheep poxvirus P32 gene, nodular skin disease virus P32 gene, sheep sore virus VIR gene, monkeypox virus and cowpox virus F3L gene as positive controls, which are used for chip positive controls;

[0058] 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.

[0059] 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 .

[0060] 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 the corresponding allele 1, FAM fluorescence (red) is displayed, and if the template DNA contains allele 2, HEX fluorescence (bright green) is displayed.

[0061] 2. Preparation of the kit

[0062] 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.

[0063] PCR amplification reagent: KASP TF V4.0 2×Master mix (LGC, catalog number KBS-1016-002).

[0064] The control substance is a recombinant plasmid of goatpox virus P32 gene, sheeppox virus P32 gene, lumpy skin disease virus P32 gene, monkeypox virus F3L gene, cowpox virus F3L gene, and sheep sore virus VIR gene, which were constructed and confirmed by sequencing.

[0065] 3. Experimental methods of the kit

[0066] (1) Extracting genomic DNA from the sample to be tested (blood, blister fluid, swab, etc.);

[0067] (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.

[0068] (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.

[0069] (4) After completing step 3, the chip is scanned and the data is analyzed. The chip reading principles and standards are shown in Table 1.

[0070] Table 1 Chip interpretation principles

[0071]

[0072] Example 3: Validation of the kit using simulated samples

[0073] The kit prepared in Example 2 was verified by selectively combining the constructed standard plasmid and the relevant host genomic DNA into simulated samples 1 to 14, and using TE Buffer as the negative sample 15 (see Table 2).

[0074] Table 2 Test sample information and chip test results

[0075]

[0076]

[0077] Anticoagulated blood was collected from healthy cattle, sheep, goats and humans, and genomic DNA was extracted from cattle, sheep, goats and humans using the instructions of MiniBEST Whole Blood Genomic DNA Extraction Kit (TaKaRa, Cat. No. 9781). The OD260nm / OD280nm ratio and concentration of the recombinant plasmid were measured using a spectrophotometer. The positive plasmid copy number (copy / μL) = (plasmid concentration × 10 -9 × dilution factor × 6.02 × 10 23 ) / (660 Daltons / base × number of bases) to calculate the copy number of the recombinant plasmid and use it as the positive standard for fluorescent quantitative PCR.

[0078] According to the instructions of Mini BEST Viral RNA / DNA Extraction Kit (TaKaRa, Cat. No.: 9766), genomic DNA of GTPV, SPPV, LSDV and ORFV reference strains was extracted, and the target gene was amplified by PCR. According to the instructions of Mini BEST Agarose Gel DNA Extraction Kit (TaKaRa, Cat. No.: 9762), the PCR product was recovered and purified, cloned into the pMD19-T vector, and transformed into Dh5α competent cells. After correct sequencing, plasmid DNA was extracted using Mini BEST Plasmid Purification Kit (TaKaRa, Cat. No.: 9760) to prepare recombinant plasmid standards. According to the reference sequences of monkeypox virus and vaccinia virus F3L genes registered in GenBank, we commissioned Sangon Biotech (Shanghai) Co., Ltd. to perform artificial synthesis, and then constructed into pMD19-T vectors, transformed into Dh5α competent cells, and after correct sequencing, used MiniBEST Plasmid Purification Kit (TaKaRa, Cat. No.: 9760) to extract plasmid DNA and prepare recombinant plasmid standards. The OD260nm / OD280nm ratio and concentration of the recombinant plasmid were measured by spectrophotometer, and the recombinant plasmid copy number (copy / μL) was calculated according to the formula: (plasmid concentration × 10-9 × dilution × 6.02 × 1023) / (660 Daltons / base × number of bases) and used as the positive standard for fluorescent quantitative PCR.

[0079] The extracted host genomic DNA and standard plasmid were selectively combined according to the infected host and named sample 1 to sample 14. In addition, the TE Buffer provided by Mini BEST Viral RNA / DNA Extraction Kit (TaKaRa, Cat. No. 9766) was used as a negative control and named sample 15.

[0080] Samples 1 to 14 (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).

[0081] Results Figure 2 The results showed that the method and kit of the present invention can specifically detect goatpox virus, sheeppox virus, lumpy skin disease virus, monkeypox virus, cowpox virus and sheep sore virus standard plasmids, and have the potential to simultaneously detect the above six different pox viruses.

[0082] Example 3 Detection of clinical samples

[0083] The kit prepared in Example 2 was used and operated according to the method of use to test 52 clinical samples. Among the 52 clinical samples, 4 were collected from whole blood samples of healthy cattle, sheep, goats and humans, and genomic DNA was extracted, which were named cattle genomic DNA, sheep genomic DNA, goat genomic DNA and human genomic DNA respectively; the remaining 48 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 showed that the test results of the 48 clinical samples were 100% consistent with the PCR test and sequencing test results.

[0084] Table 3 Clinical sample information and chip test results

[0085]

[0086]

[0087] 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 6 poxviruses, wherein the 6 poxviruses include goat pox virus, sheep pox virus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep pox virus; characterized in that: The primer combination includes primer sets 1-5; The primer set 1 is used for differential diagnosis of sheeppox virus and lumpish dermatosis 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 lumpish dermatosis virus, and its nucleotide sequence is shown in SEQ ID NO.7-9; the primer set 4 is used for universal detection of monkeypox virus and cowpox virus, and its nucleotide sequence is shown in SEQ ID NO.10-11; the primer set 5 is used for detection of sheep pox virus, and its nucleotide sequence is shown in SEQ ID NO.12-13.

2. The primer combination according to claim 1, characterized in that The primer combination also includes internal control primer group 1, internal control primer group 2 and internal control primer group 3; 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.14-16; 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.17-19; the internal control primer group 3 is used to detect the conserved region of the human genome sequence, and its nucleotide sequence is shown in SEQ ID NO.20-22.

3. Use of the primer combination according to claim 1 or 2 in the preparation of a product for the simultaneous detection of six poxviruses, wherein the six poxviruses include goatpox virus, sheeppox virus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep pox 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 substance is a recombinant plasmid of goatpox virus P32 gene, sheeppox virus P32 gene, nodular skin disease virus P32 gene, monkeypox virus and cowpox virus F3L gene, and sheep sore virus VIR gene fragment.

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 sample genome DNA extraction reagent, a PCR amplification reagent, and a KASP reagent.

8. A method for detecting the presence of six poxviruses for non-disease diagnosis purposes, wherein the six poxviruses include goat poxvirus, sheep poxvirus, lumpish skin disease virus, monkeypox virus, cowpox virus and sheep pox virus; characterized in that: The method comprises the following steps: (1) Extracting genomic DNA from the sample to be tested; (2) respectively mixing the genomic 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 claim 4 or the detection chip of the kit described in 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 the step (2), the volume ratio of genomic 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 the 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 / rash, nodules, scabs, lymph nodes, lung / semen, urine, and milk; and any one or more of human blister fluid, eye secretion swabs, oropharyngeal secretion swabs, nasopharyngeal secretion swabs, whole blood, plasma, serum, papules / rash, nodules, and scabs.

Images