Kit for detecting orf virus of sheep and detection method thereof

A technology for pustular dermatitis and detection methods, applied in biochemical equipment and methods, microbial measurement/testing, fluorescence/phosphorescence, etc. Complicated problems, to achieve the effect of simple operation, low cost and high specificity

Active Publication Date: 2013-08-21
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From an economic point of view, the price of foreign testing reagents for testing a sample is often tens of yuan or even hundreds of yuan, which is too high for sheep farmers to accept
At present, there is only a common PCR kit for the detection of sheep infectious pyoderma virus. The operation of this kit is relatively complicated, and the results must be determined by electrophoresis strips, which is time-consuming and laborious, and the ethidium bromide added to the gel during electrophoresis is harmful to human health. , take extra care when operating

Method used

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  • Kit for detecting orf virus of sheep and detection method thereof
  • Kit for detecting orf virus of sheep and detection method thereof
  • Kit for detecting orf virus of sheep and detection method thereof

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Embodiment Construction

[0029] The invention provides a detection kit for sheep infectious pustular dermatitis virus, which consists of the following components:

[0030] (1) Premix Ex Taq, purchased from Bao Biological Engineering (Dalian) Co., Ltd., model RR390Q.

[0031] (2) Positive control

[0032] The positive control of this kit invention is a plasmid containing the H2L gene of the sheep infectious pustular dermatitis virus, and the preparation method of the plasmid containing the H2L gene of the sheep infectious pustular dermatitis virus is as follows: The H2L gene was amplified from the virus, cloned into pMD-18T, transformed into Escherichia coli competent cells JM109 competent cells, spread on LB plates containing 100mg / L ampicillin, and cultured at 37°C for 12-16 hours. After the blue-white screening, the plasmid was extracted with a plasmid (small amount) extraction kit, and the plasmid whose sequence was determined to be positive was named pMD-H2L. Its copy number concentration is 2.8...

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Abstract

The invention provides a detection method for detecting the orf virus of sheep. Real-time fluorescent quantitative PCR (polymerase chain reaction) detection is performed on a DNA (deoxyribonucleic acid) template of a sample by designing a pair of primers and a probe. The invention further provides a kit for detecting the orf virus of the sheep by utilizing the detection method. The real-time quantitative method which is established against H2L genes in a conserved region of the ORFV (orf virus) is high in specificity and good in stability. The kit for detecting the orf virus of the sheep, provided by the invention, is simple and convenient to operate, a user only needs to add DNA of the sample to be detected into a reaction tube, detection results can be obtained according to an amplification curve, the operation is time-saving and labor-saving, the cost is lower, and the time is shortened from original 3-4 hours to 1-2 hours; and furthermore, the sensitivity is higher than that of the ordinary PCR, and the detection method and the kit can be used for detecting the low content of the orf virus of the sheep, can be suitable for all levels of prevention and control units, basic-level veterinary stations, large and medium-sized breeding farms and the like in China, and has better application prospects.

Description

technical field [0001] The invention relates to a TaqMan Real-time kit for detecting sheep infectious pustular dermatitis virus and a detection method thereof, in particular to a kit and a detection method for detecting sheep infectious pustular dermatitis virus by using TaqMan real-time fluorescent quantitative PCR. Background technique [0002] Orf Virus (ORFV) is referred to as Orf Virus (ORFV), which can cause contact infection between Ovis animals and humans, and even cause problems in the development of the sheep industry and environmental safety. The ORFV-contaminated environment has a very high risk of infection for young sheep and lactating ewes. They mainly infect the skin or mucous membrane of the host, and can lead to severe persistent infection and secondary infection. ORFV has distinct species characteristics and rich functional genes. Its genome is a double-stranded DNA with closed ends, about 138kb in length, with a core region (88kb) in the center and invert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 刘湘涛田宏吴锦艳陈妍尚佑军张克山尹双辉王光祥杨顺利刘永杰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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