Capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method

A technology of real-time fluorescence quantification and sheep pox virus, which is applied in the field of animal virus molecular biology inspection, can solve the problem of no inactivation effect, and achieve the effect of simple identification

Active Publication Date: 2014-11-19
重庆海关技术中心 +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Repeated freezing and thawing had no obvious inactivation effect
However, there are no reports on the kits and detection meth

Method used

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  • Capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method
  • Capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method
  • Capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, design and screening of primers

[0043] The primers and probes for fluorescent quantitative PCR amplification of three kinds of capipoxviruses were designed according to the reference sequences of bovine pimple skin disease virus, goat poxvirus and sheep poxvirus published by GenBank, and carried out with MEGA5. Align and analyze the sequences and design primers and probes in the conserved regions. The probe design software Primer Express 3.0 was used to design 3 sets of fluorescent quantitative primers, which were synthesized by Yingjun (Shanghai) Co., Ltd. ABI 7500 FAST PCR instrument was used to monitor the amplification in the reaction process in real time. Analyze parameters such as the starting time of amplification, the time to enter the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau, and screen out a group of fluorescent quantitative PCR amplification with the highest amplification rate and go...

Embodiment 2

[0051] Embodiment 2, the preparation of positive control substance

[0052] Preparation of bovine pimple skin disease virus positive plasmid: use a kit to extract the nucleic acid of the bovine pimple skin disease virus cell culture, carry out PCR and electrophoresis identification of the nucleic acid, and use PCR upstream primer SEQ ID NO.6 and PCR downstream primer SEQ ID NO .7 Carry out amplification, and recover the amplified band using the gel extraction kit. According to the ratio of 1:10 and the PMD19T carrier for ligation reaction, ligated overnight at 4°C, transformed into DH5α bacteria, after resistance selection and PCR identification positive, re-sequencing verification, using a spectrophotometer to measure the OD value of its nucleic acid to make it 260 The ratio of / 280 is between 1.8 and 2.0. The preparation method of goat pox virus positive plasmid and sheep pox virus positive plasmid is the same as that of bovine pimple skin disease virus positive plasmid.

Embodiment 3

[0053] Embodiment 3, the preparation of negative control substance

[0054] The kit was used to extract the DNA of the epithelial tissue without the infection of the genus poxvirus, and carry out PCR and electrophoresis identification.

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Abstract

The invention discloses a capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, a kit and a detection method. The multiple Taqman-MGB real-time fluorescence quantitative PCR detection method can be used for rapid detection of Lumpy Skin Disease Virus (LSDV), Goatpox Virus (GTPV) and Sheeppox Virus (SPPV). According to the method, on the basis of conserved domains of target sequences of the LSDV, the GTPV and the SPPV, a pair of primer and three Taqman-MGB probes are designed. The method only needs two step amplification method and simple reaction conditions for fast, efficient, specific and highly-sensitive detection of an objective target sequence, is simple in operation, does not need expensive equipment and reagents, has no technical requirement on operators, is low in detection cost and short in testing time, and can avoid cross contamination caused by agarose electrophoresis so as to improve the detection accuracy and reliability.

Description

technical field [0001] The invention relates to the field of animal virus molecular biology testing methods, in particular to a primer, method and kit for multiplex real-time fluorescence quantitative PCR detection of capipoxvirus Taqman-MGB probes. Background technique [0002] Capripoxvirus (CPV) includes Goatpox Virus (GTPV), Sheep Pox Virus (SPPV) and Lumpy Skin Disease Virus (LSDV), which can cause goats, Extensive nodules and edema on the skin and organ surfaces of sheep and cattle. The milk production of sick animals is drastically reduced, and the quality of fur is greatly reduced, causing huge economic losses. Goat pox, sheep pox caused by sheep pox virus, also known as sheep "smallpox", is an acute, febrile, contact infectious disease of sheep. Sheep pox and goat pox are listed as a first-class infectious disease in my country, which has a great impact on animal husbandry. According to the difference in virulence of different strains, the lethality rate of suscep...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2561/101C12Q2545/101
Inventor 聂福平王昱杨俊李应国王国民李贤良
Owner 重庆海关技术中心
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