Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2

A real-time fluorescence quantitative and porcine circovirus technology, applied in the field of molecular biology, achieves high sensitivity, improved sensitivity, and simple detection methods

Active Publication Date: 2012-03-21
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on the specific binding of the TaqMan probe to the target fragment, the increase in the amount of fluorescence monitored during the whole process represents the generation of the target fragment, ensuring the specificity of the experiment to solve the problem of rapid and accurate detection of porcine circovirus type 2 question

Method used

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  • Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2

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Embodiment 1

[0037] 1) Obtaining the target fragment

[0038] In the detection study of porcine circovirus type 2, genomic DNA was used as template for PCR amplification. The PCR reaction system was 25 μl: 12.5 μl of 2×PCR Mix, 1 μl of upstream and downstream primers, 2 μl of genomic DNA, and added deionized water to a total volume of 25 μl. The PCR reaction program is: 94°C for 5min, 94°C for 1min, 52°C for 1min, 72°C for 1min, 72°C for 10min, 35 cycles. The PCR product is recovered by agarose gel electrophoresis, and the PCR product is purified with a gel recovery kit to obtain the target. fragment.

[0039] 2) Prepare the connection reaction system

[0040] In 2 μl of purified PCR product, 1 μl of pGEM-T easy vector, add T 4 1 μl of ligase, 5 μl of quick ligation buffer, and 1 μl of deionized water constitute a 10 μl ligation reaction system. Incubate for 1 hour at room temperature or overnight at 4°C. The connection solution was purified by column, with 20μl ddH 2 O elutes.

[004...

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Abstract

The invention discloses a real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for a porcine circovirus type 2, which includes the steps: 1 designing and synthesizing specific primers and probes, 2 obtaining target fragments by preparing a PCR reaction system and reacting according to PCR reaction procedures, 3 preparing ligation reaction liquid, 4 converting and extracting plasmid templates, 5 performing qualitative PCR detection, and 6 performing quantitative PCR detection. The detection method is simple, rapid, high in sensitiveness, high in specialty, fine in repeatability and extremely low in quantitative detection limit and detection limit, and the porcine circovirus type 2 can be detected quantitatively and accurately by the aid of the high sensitiveness, so that epidemic of the porcine circovirus type 2 can be prevented and controlled.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a real-time fluorescent quantitative PCR detection method for porcine circovirus type 2. Background technique [0002] At present, methods commonly used to detect viruses include enzyme-linked immunosorbent assay (ELISA), qualitative PCR technology and real-time fluorescent quantitative PCR technology. ELISA is highly sensitive, but the test results may include false positives. Qualitative PCR technology is low in cost and easy to operate, but its sensitivity is relatively low, and because its amplification products need to be analyzed by gel electrophoresis, it is easy to produce pollution. Real-time fluorescent quantitative PCR technology is widely used because of its high sensitivity, high specificity and easy operation. [0003] Real-time fluorescent quantitative PCR technology is a method that adds fluorescent groups to the PCR system, uses the accumulation of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 赵凯唐雪明韩芳婷邹勇李春华吴潇朱宏谭芙蓉王金斌蒋玲曦陶世如刘启文施伟
Owner SHANGHAI ACAD OF AGRI SCI
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