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Quantitative PCR detection method of minute virus of mice and primers and probe of quantitative PCR detection method

A parvovirus and detection method technology, applied in the biological field, can solve the problems of long detection time, complicated operation, poor specificity and stability, etc., and achieve the effects of short detection time, simple operation, high specificity and sensitivity

Inactive Publication Date: 2016-04-20
SUZHOU YAOMING KANGDE INSPECTION TESTING +1
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Problems solved by technology

[0004] At present, the commonly used methods for detecting MVM include enzyme-linked immunosorbent assay, immunoenzyme assay and immunofluorescence assay, but these methods have disadvantages such as complicated operation, long detection time, poor specificity and stability.

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  • Quantitative PCR detection method of minute virus of mice and primers and probe of quantitative PCR detection method
  • Quantitative PCR detection method of minute virus of mice and primers and probe of quantitative PCR detection method

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Embodiment Construction

[0019] In order to have a more specific understanding of the technical content, characteristics and effects of the present invention, now in conjunction with specific embodiments, the present invention is described in detail as follows:

[0020] The quantitative PCR detection method of the mouse parvovirus of the present embodiment, the specific steps are:

[0021] Step 1, use proteinase K / SDS digestion, multiple phenol / chloroform extraction, ethanol precipitation methods, or use other commercial kits to extract the DNA of the sample to be tested, and adjust the concentration of the DNA template to 0.1 μg / μL . If the DNA concentration is very low, resuspend the DNA in a minimal volume of 1X TE buffer.

[0022] Step 2: Prepare a PCR reaction solution containing 1×Mastermix, upstream primers (final concentration: 100 nM), downstream primers (final concentration: 100 nM), and probes (final concentration: 250 nM). The sequences of the primers and probes used are as follows:

[...

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Abstract

The invention discloses a quantitative PCR detection method of the minute virus of mice. The method includes the steps of firstly, extracting DNA of a to-be-detected sample; secondly, preparing PCR reaction liquid containing Master mix, the upstream primer, the downstream primer and the probe; thirdly, diluting a standard substance in a gradient mode; fourthly, loading the negative control of reagents, the negative control of the to-be-detected sample and the to-be-detected sample where the standard substance is added; fifthly, conducting the PCR reaction. The invention further discloses sequences of the primer pair and the probe which are used in the method. The minute virus of mice is detected through the quantitative PCR method using the Taq Man probe; compared with a traditional MVM detection method, high specificity and sensitivity are achieved, and the method is simple, easy to operate, small in detection time consumption, good in result stability and repeatability and suitable for MVM infection test of all stages in the biological production process.

Description

technical field [0001] The invention relates to the field of biology, in particular to quantitative PCR detection of mouse parvovirus, primers and probes thereof. Background technique [0002] With the rapid development of biomedical technology, people pay more and more attention to the safety of biological products such as vaccines, and strengthen their quality control. The quality control of biological products is not only the quality control of the final product, but also the quality control of the whole production process. Therefore, regulatory authorities around the world require manufacturers to pass complex safety tests at all stages of the production process to prove the consistency, stability, and purity of the product before clinical trials are approved and before the final product of biopharmaceuticals and vaccines is released. [0003] Rodents and the derived cell line CHO are widely used in the production of vaccines and therapeutic biological products. CHO ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/113C12Q2561/101
Inventor 钱学敏姜东秀刘大钧
Owner SUZHOU YAOMING KANGDE INSPECTION TESTING
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