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Dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus

A goat pox virus, sheep pox virus technology, applied in the field of probes, double fluorescent PCR primers, methods and kits, can solve the problem of inability to distinguish sheep pox virus and bovine nodular skin disease virus, unable to diagnose in time, and ineffective. Distinguish and other problems to achieve the effect of improving detection accuracy and precision, high accuracy and strong specificity

Active Publication Date: 2020-12-25
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no diagnostic reagents approved for marketing for the diagnosis of goatpox virus and bovine nodular skin disease virus. Goat pox virus and bovine nodular skin disease virus
Therefore, there is no effective way to distinguish the goat pox vaccine strain from the wild strain of bovine nodular skin disease virus, and the inability to make a timely diagnosis is also an important reason for the spread of the disease

Method used

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  • Dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus
  • Dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus
  • Dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This set of primers and probes is designed for all members of the genus Goatpoxvirus. Compared with the gene sequences of a large number of Goatpoxviruses, Goatpoxviruses, and Bovine Nodular Skin Disease Viruses recorded on GenBank, it is found that the open reading frame in the sequence of the virus genus The ORF130 gene sequence is highly conserved. The conserved region in the ORF130 gene sequence was determined by sequence comparison, and primers and probes were designed for this region to detect viruses in the genus Capapoxvirus. After a lot of design and screening, CaPV-130-F2 and CaPV-130-R2 are upstream and downstream primers, and CaPV-130-P2 is a probe. The nucleotide sequences of the primer set and probe are:

[0047] CaPV-130-F2:5'-TGGAAGCAAGTGRTAACGK-3' (SEQ ID NO: 1);

[0048] CaPV-130-R2:5'-CTATTCTATCCCATCATTATACGTA-3' (SEQ ID NO:2);

[0049] CaPV-130-P2:5'-TCATCAAAAGGAAACGGATC-3' (SEQ ID NO:3).

[0050] The fluorescent group labeled at the 5' end of CaPV...

Embodiment 2

[0057] (1) Artificially synthesize the target gene sequence of cappoxvirus (Genbank accession number: KX576657) and bovine nodular skin disease virus target gene sequence (Genbank accession number: MN072619), and the genes at both ends are connected to the vector, and the recombined Carrier DNA was used as a positive plasmid for PCR amplification.

[0058] (2) With the positive plasmid prepared, use embodiment 1 primer set CaPV-130-F2, CaPV-130-R2 and LSD-132-F5, LSD-132-R6, probe CaPV-130-P2 and LSD-132 -LP5 performs fluorescent PCR amplification on the diluted plasmid standard under optimal amplification conditions. The amplification system is:

[0059]

[0060]

[0061] The amplification program is: 50°C, 3min; 95°C pre-denaturation for 5min; 95°C denaturation for 15s, 58°C annealing and extension for 30s, a total of 40 cycles, collecting fluorescent signals, selecting goat poxvirus, selecting FAM channel and bovine nodular skin disease Select the VIC channel to obta...

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Abstract

The invention discloses dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus. The kit comprises two pairs of specific primers CaPV-130-F2 and CaPV-130-R2, and LSD-132-F5 and LSD-132-R6, and TaqMan-MGB probes, wherein 5'-end labeled report fluorescent dyes of CaPV-130-P2 and LSD-132-LP5 probes are FAM and VIC respectively, and 3'-end labeled fluorescence quenching groups are MGB. The kit provided by the invention can be used for detecting goat pox virus, sheep pox virus and lumpy skin disease virus in scabs, papules, vesicles, blood and other samples of sheep, goats and cattle, and can provide certain technical support and help for the diagnosis, epidemiological investigation, prevention and control and purification of capripoxvirus.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a dual fluorescent PCR primer, probe, method and kit for differentiating sheeppox virus (genus Goatpoxvirus) and bovine nodular skin disease virus. Background technique [0002] The genus Capripoxvirus (Capripoxvirus) belongs to the family Poxviridae and the subfamily Chordatepoxviridae, which includes goat poxvirus (GTPV), sheep poxvirus (SPPV) and bovine nodular skin disease virus (LSDV). A DNA virus with a genome length of about 150kb, encoding 147 open reading frames. [0003] The disease symptoms caused by goat pox virus are mainly skin papule-pustular pox. Goat pox can infect goats of all breeds, sexes and ages, among which lambs are the most susceptible, with an infection rate of 100%. It infects other goats mainly through contact with damaged skin, respiratory inhalation or vector transmission, and spreads rapidly among the same group. Goat pox virus has a hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2561/101
Inventor 翟少伦娄亚坤吕殿红翟颀温肖会杨楠杨丹芳
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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