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Lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method

A real-time fluorescence quantification technology for goitre skin disease, which is applied in the field of animal virus molecular biology testing, and achieves the effects of simple instrument, low technical quality requirements, and simple identification.

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

AI Technical Summary

Problems solved by technology

The above reports indicate that there is also a risk of LSD outbreaks in other regions outside of Africa
However, there is no report on the kit and detection method for detecting bovine pimple skin disease virus by using TaqMan-MGB probe fluorescent quantitative PCR amplification technology at present

Method used

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  • Lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method
  • Lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method
  • Lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, design and screening of primers

[0035] Bovine pimple skin disease virus fluorescent quantitative PCR amplification primers and probes are designed based on the reference sequence of bovine pimple skin disease virus published by GenBank, compared with MEGA5, sequence analysis and primers and probes in its conserved regions design. The probe design software Primer Express 3.0 was used to design 2 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, and the amplification of different primer sets The parameters such as the starting time of starting time, the time of entering the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau stage were analyzed, and a set of fluorescent quantitative PCR amplification primers with the highest amplification rate and good speci...

Embodiment 2

[0041] Embodiment 2, the preparation of positive control substance

[0042] Use the kit to extract the nucleic acid of bovine pimple skin disease virus cell culture, identify the nucleic acid by PCR and electrophoresis, use PCR upstream primer SEQ ID NO.4 and PCR downstream primer SEQ ID NO.5 to amplify, and use gel to recover The kit recovers the amplified band. According to the ratio of 1:10 and the PMD19-T 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, so that Its 260 / 280 ratio is between 1.8 and 2.0.

Embodiment 3

[0043] Embodiment 3, the preparation of negative control substance

[0044] The kit was used to extract the DNA of bovine tissue without bovine pimple skin disease virus infection, and carry out PCR and electrophoresis identification.

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Abstract

The invention discloses a lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, a kit and a detection method. According to the method, 2 pieces of the primers and a probe are designed according to one region of a target sequence, the kit includes 2* PremixExTaqTM buffer, a positive control, a negative control and sterilized deionized water. 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, and is low in detection cost and short in testing time.

Description

technical field [0001] The invention relates to the field of animal virus molecular biology detection methods, in particular to a primer, a kit and a detection method for real-time fluorescent quantitative PCR detection of bovine pimple skin disease virus Taqman-MGB probe. Background technique [0002] Lumpy skin disease (LSD), also known as bovine nodular rash or bovine nodular skin disease, is an acute, sub All kinds of cattle are susceptible to acute infectious diseases, and are listed as animal diseases that must be notified by the World Organization for Animal Health (OIE). The infection rate of the disease is 5% to 85%, and the fatality rate is 10%. 50%. At the same time, it can also cause primary and secondary pneumonia. The limbs of infected animals suffer from lameness due to synovitis and tenosynovitis, abortion of sick female animals, temporary or permanent infertility of male animals, and death in severe cases; Depression or hollows cause huge economic losses,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2561/101C12Q2545/101
Inventor 聂福平杨俊王昱李应国王国民李贤良
Owner 重庆海关技术中心
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