Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of goat poxvirus taqman-mgb probe

A technology of real-time fluorescence quantification and goat pox virus, which is applied in biochemical equipment and methods, methods based on microorganisms, microbiological determination/inspection, etc., can solve the problem that there is no detection method for identifying goat pox, inability to distinguish virus detection, and time-consuming To achieve high-throughput rapid detection, overcome long detection time, and reduce costs

Active Publication Date: 2016-04-13
重庆海关技术中心
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AI Technical Summary

Problems solved by technology

However, this technique requires purification and 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 cannot distinguish between these two viruses, so inventing a method that can identify and detect goatpox virus is of great significance to the research of these two viruses and the virus of the genus Goatpox
In terms of detection of goat pox virus, there is no detection method using TaqMan-MGB fluorescence quantitative method to identify goat pox
But, there is no report on the kit and detection method of detecting goat pox virus by using TaqMan-MGB probe fluorescent quantitative PCR amplification technology at present

Method used

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  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of goat poxvirus taqman-mgb probe
  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of goat poxvirus taqman-mgb probe
  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of goat poxvirus taqman-mgb probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, design and screening of primers

[0034]Goat poxvirus fluorescent quantitative PCR amplification primers and probes were designed based on the goat poxvirus reference sequence published by GenBank, compared with MEGA5, the sequences were analyzed, and primers and probes were designed in the conserved regions. The probe design software PrimerExpress3.0 was used to design 4 sets of fluorescent quantitative primers, which were synthesized by Yingjun (Shanghai) Co., Ltd., and the ABI7500FASTPCR instrument was used to monitor the amplification in the reaction process in real time. Time, the time to enter the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau were analyzed, and a set of fluorescent quantitative PCR amplification primers with the highest amplification rate and good specificity were screened out, which were respectively marked as SEQIDNO .1~SEQ ID NO.3. Wherein primer upstream in primer group: SE...

Embodiment 2

[0040] Embodiment 2, the preparation of positive control substance

[0041] Use the kit to extract the nucleic acid of the goat pox virus cell culture, identify the nucleic acid by PCR and electrophoresis, use PCR upstream primer SEQIDNO.4 and PCR downstream primer SEQIDNO.5 to amplify, and use the gel recovery kit to recover and amplify of strips. 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

[0042] Embodiment 3, the preparation of negative control substance

[0043] The kit was used to extract the DNA of sheep tissue without goatpox virus infection, and then identified by PCR and electrophoresis.

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Abstract

The invention discloses a goat pox virus 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, 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 epidemic molecular biology testing methods and testing reagents, in particular to a primer, a kit and a testing method for goat pox virus Taqman-MGB probe real-time fluorescent quantitative PCR testing. Background technique [0002] Goat pox virus (GTPV) belongs to the Capripoxvirus genus (Capripoxvirus) virus, which can cause goat pox in animals. The main symptoms of affected sheep are fever, extensive papules or nodules on the skin, mucous membranes, and organ surfaces, and enlarged lymph nodes. Skin edema, infected animals become emaciated, milk production is greatly reduced, and in severe cases, death is caused, which brings great harm to the breeding industry and causes serious economic losses. Goat pox and sheep pox are collectively called "sheep pox", which can infect humans, so it is of great significance in public health (KeySJ., 2007). Some isolated strains of goat pox virus and sheep pox virus ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 王昱聂福平杨俊王国民李应国
Owner 重庆海关技术中心
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