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Primer and kit for real-time fluorescence quantification PCR detection of wild strain TaqMan-MGB of cattle nodular skin disease virus and detection method

A real-time fluorescence quantitative and nodular technology, which is applied in the field of animal virus molecular biology detection, can solve the problems of high risk of LSDV and achieve high-efficiency amplification, simple identification results, and high sensitivity

Inactive Publication Date: 2018-12-07
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above reports show that the risk of LSDV being introduced into my country is extremely high

Method used

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  • Primer and kit for real-time fluorescence quantification PCR detection of wild strain TaqMan-MGB of cattle nodular skin disease virus and detection method
  • Primer and kit for real-time fluorescence quantification PCR detection of wild strain TaqMan-MGB of cattle nodular skin disease virus and detection method
  • Primer and kit for real-time fluorescence quantification PCR detection of wild strain TaqMan-MGB of cattle nodular skin disease virus and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, design and screening of primers

[0034] According to the comparison and analysis of the reference sequence of the bovine nodular skin disease virus wild strain and the vaccine strain sequence published on the Genbank website, based on the advantages of the TaqMan MGB probe, it is possible to identify a mutated gene of 1 base and analyze the LSDV wild virus in depth Finally, the ORF126 region of LSDV was selected, and its specific primers and TaqManMGB probes were innovatively designed using Primer Premier 5.0 biological software, which were respectively marked as SEQ ID NO.1-SEQ ID NO.3 . All primers and probes were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for synthesis. Among them, the upstream primer of bovine nodular skin disease virus wild strain in the primer set: SEQ ID NO.1; the downstream primer of bovine nodular skin disease virus wild strain: SEQ ID NO.2; bovine nodular skin disease virus wild strain Strain probe: SEQ ID NO.3. The c...

Embodiment 2

[0042] Embodiment 2, positive recombinant plasmid construction

[0043] The artificially synthesized LSDV field strain genome was amplified by PCR and identified by electrophoresis. PCR upstream primer SEQ ID NO.4 and PCR downstream primer SEQ ID NO.5 were used for amplification, and the amplified target band was recovered using a gel recovery kit. Carry out ligation reaction with pMD19-T vector at a ratio of 1:10, ligate overnight at 4°C, transform DH5a bacteria, after resistance selection and PCR identification positive, re-sequencing verification, use a spectrophotometer to measure the OD value of its nucleic acid, so that Its 260 / 280 ratio is between 1.8 and 2.0.

Embodiment 3

[0044] Embodiment 3, the preparation of negative control substance

[0045] DNA from bovine nodular skin disease virus-infected bovine tissue was extracted with a commercial kit, and PCR was negative.

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Abstract

The invention discloses a primer and kit for the real-time fluorescence quantification PCR detection of a wild strain TaqMan-MGB of a cattle nodular skin disease virus and a detection method. According to the method, two primers and a specific MGB probe are designed and respectively have the DNA sequences of SEQ ID NOs.1-3. The kit contains amplification reaction liquid, a positive control, a negative control and sterilized deionized water. A target sequence can be rapidly, efficiently, specifically and sensitively detected by virtue of a two-step amplification method, the operation is simpleand convenient, expensive instruments and reagents are not used, the technical requirements on operators are not required, the detection cost is low, the flux is high, and the detection period is short.

Description

technical field [0001] The invention relates to the field of animal virus molecular biology detection methods. Specifically relates to a primer, a kit and a detection method for real-time fluorescent quantitative PCR detection of bovine nodular skin disease virus wild strain TaqMan-MGB. Background technique [0002] Bovine nodular skin disease (Lumpy skin disease, LSD), also known as bovine bump skin disease, bovine nodular rash or bovine nodular dermatitis, is caused by bovine nodular skin disease virus (Lumpy skin disease virus, LSDV) It is an acute and subacute infectious disease of cattle. All kinds of cattle are susceptible to it. It is listed as an animal infectious disease that must be notified by the World Organization for Animal Health (OIE). class of infectious diseases. The infection rate of the disease is 5%~85%, and the case fatality rate is 10%. Its main clinical features are a large number of pimple-like nodules on the skin surface of sick cows, body tempera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 聂福平王昱杨俊李贤良王国民李应国
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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