Goat pox virus and sheep pox virus dual-PCR (Polymerase Chain Reaction) detection kit and detection method

A technology of sheep pox virus and goat pox virus, which is applied in biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc., can solve the problem that there are no multiple detection methods for goat pox and sheep pox, and the inability to distinguish detection Viruses, time-consuming and labor-intensive problems, etc., to achieve high sensitivity, low false positive rate, and high sensitivity

Active Publication Date: 2012-03-07
重庆海关技术中心 +1
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  • Application Information

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Problems solved by technology

However, this technique requires purification and enzyme digestion of the product after the polymerase chain reaction, which is time-consuming and laborious.
At present, some other detection techniques at home and abroad can not distinguish between these two viruses, so a detection method that can detect goat pox virus and sheep pox virus simultaneously is invented, wh

Method used

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  • Goat pox virus and sheep pox virus dual-PCR (Polymerase Chain Reaction) detection kit and detection method
  • Goat pox virus and sheep pox virus dual-PCR (Polymerase Chain Reaction) detection kit and detection method
  • Goat pox virus and sheep pox virus dual-PCR (Polymerase Chain Reaction) detection kit and detection method

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Embodiment 1

[0040] Embodiment 1, design and screening of primers of the present invention

[0041] Based on the known goat poxvirus nucleic acid sequence and the full-length genome sequence of the sheep poxvirus nucleic acid sequence, multiple alignments were performed using ClustW software, and specific primers were designed with Primer Premier 5.0 software, respectively marked as: SEQ ID NO1...SEQ ID NO4. All primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd. The goat pox virus and the sheep pox virus cytotoxic DNA are respectively extracted with a nucleic acid extraction kit, respectively amplified by the primers designed in claim 1, and primers with non-specific amplification are excluded.

[0042] Get primers:

[0043]

Embodiment 2

[0044] Embodiment 2, the preparation of positive control substance

[0045]Commercially available commercial kits were used to extract DNA from positive goat pox virus and sheep pox virus cell cultures, and the DNA of goat pox virus and sheep pox virus were amplified respectively with Mix PCR reaction tubes. The reactions could amplify a 177 bp and A band around 222 bp. A gel recovery kit was used to recover the amplified target band, and a spectrophotometer was used to measure the concentration of the recovered nucleic acid. Carry out ligation reaction with pMD19-T vector respectively, ligate overnight at 4°C, transform the ligated products into JM109 competent cells, resistance screening, shaking bacteria, PCR identification positive, extract the recombinant plasmid, sequence verification, and obtain the target fragment For the recombinant plasmids pMD19-T-G and pMD19-T-S, the positive plasmids of the two viruses were mixed in equal proportions, aliquoted in 50 μL each, and...

Embodiment 3

[0046] Embodiment 3, the preparation of negative control substance

[0047] The DNA of goat or sheep skin or muscle tissue free from goat pox virus and sheep pox virus infection was extracted using a commercially available nucleic acid extraction kit. Dilute with sterilized deionized water, control the concentration at 80-100ng / μL, and pack into 50μL each.

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Abstract

The invention discloses a goat pox virus and sheep pox virus dual-PCR (Polymerase Chain Reaction) detection kit and a dual-PCR method for rapidly identifying and detecting goat pox virus and sheep pox virus. One pair of PCR primers is respectively designed according to genome sequences of the goat pox virus and the sheep pox virus, and corresponding specific fragments can be obtained through amplification, thus detection and identification of the goat pox virus and the sheep pox virus are realized. By using the dual-PCR technology, the goat pox virus and the sheep pox virus can be simultaneously detected by using a single tube, thus detection cost and workload of goat pox and sheep pox are reduced, and identification detection of the goat pox and the sheep pox is realized. According to the invention, a dual-PCR method with the advantages of strong specificity, high sensitivity, time and labor saving and easiness in observing results is established, and the advantages of convenience for operation, strong specificity, high sensitivity, high repeatability and no complex post-treatment are achieved.

Description

technical field [0001] The invention relates to a goat pox virus and a sheep pox virus kit, which belongs to the field of animal epidemic molecular biology detection methods and detection reagents, in particular to a double PCR detection method for simultaneous detection of goat pox virus and sheep pox virus in the genus Goat Pox virus Kit and application kit Biological detection method for simultaneous double PCR detection of goat pox virus and sheep pox virus. Background technique [0002] Goat pox virus (Goatpox virus) and sheep pox virus ((Sheeppox virus) both belong to the genus Capripoxvirus (Capripoxvirus), are two pathogens that cause sheep pox in animals. The main manifestations of sick sheep are fever, skin, mucous membranes, and organ surfaces. Extensive papules or nodules, swollen lymph nodes, skin edema, infected animals become emaciated, milk production is greatly reduced, and severe cases lead to death, which brings great harm to the breeding industry and caus...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 李应国聂福平肖雯杨俊肖进文王国民李贤良王昱
Owner 重庆海关技术中心
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