Primer group for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of porcine muscle regulatory factor and application of primer group

A technology of regulating factors and primer sets, applied in the field of biology

Pending Publication Date: 2020-10-27
CHINA MEAT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of real-time fluorescent quantitative reverse transcription polymerase chain reaction to detect the expression of muscle regulatory factors in cultured meat to determine the degree of differentiation

Method used

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  • Primer group for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of porcine muscle regulatory factor and application of primer group
  • Primer group for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of porcine muscle regulatory factor and application of primer group
  • Primer group for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of porcine muscle regulatory factor and application of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Fluorescent quantitative RT-PCR method simultaneously detects the expression of MyoD and MyoG before and after muscle cell differentiation

[0052] 1. Materials:

[0053]RNA extraction kit was purchased from Qiagen. The reverse transcription kit was purchased from Takara Company. TBGreenPremix Ex TaqII is a product of Takara Company.

[0054] 2. Primer and probe design and synthesis:

[0055] Using the full-length MyoD cDNA sequence (Gen Bank accession number 407604) as a template, primers were designed, and the MyoD genomic DNA sequence was considered to select the best primers.

[0056] Detection PCR upstream primer sequence is:

[0057] 5'-CACTACAGCGGTGACTCAGA-3'(SEQ ID NO.1),

[0058] The downstream primer sequences are:

[0059] 5'-GCTGTAATAGGTGCCGTCGT-3' (SEQ ID NO.2),

[0060] Using the full-length MyoG cDNA sequence (Gen Bank accession number 497618) as a template, primers were designed, and the MyoG genomic DNA sequence was considered to select...

Embodiment 2

[0092] Example 2 Verification of the detection of MyoD by fluorescent quantitative RT-PCR

[0093] Porcine muscle stem cells cultured for 1 day and 2 days were selected, and according to the method of Example 1, only the primers shown in SEQ ID NO.1 and SEQ ID NO.2 and internal reference primers were used for experiments. The results are shown in Table 5.

[0094] Table 5 detects the repeatability test result of MyoD method:

[0095]

[0096] Compared with the internal reference, the Ct value of MYoD was increased with good reproducibility.

Embodiment 3

[0097] Example 3 Fluorescent quantitative RT-PCR method to detect the expression of MyoG

[0098] Porcine muscle stem cells were selected to be cultured for 1 day, 3 days and 5 days, and according to the method of Example 1, only the primers and internal reference primers shown in SEQ ID NO.3 and SEQ ID NO.4 were used for experiments, and the results were as follows figure 2 and shown in Table 6.

[0099] Table 6 detects the repeatability test result of MyoG method

[0100]

[0101] Compared with the internal reference, the Ct value of MYoG was increased with good reproducibility.

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Abstract

The invention discloses a primer group for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection of a porcine muscle regulatory factor and application of the primer group, and belongs to the technical field of biology. The invention provides a specific primer pair capable of detecting the porcine muscle regulatory factors MyoD and MyoG and an internal reference primer pair,cDNA is obtained by extracting RNA of a sample and performing reverse transcription, and real-time fluorescent PCR is performed by using the provided primers. And therefore, mRNA expression quantitiesof the porcine muscle regulatory factors MyoD and MyoG can be rapidly, specifically, sensitively and stably detected at the same time; the primer group can be used for detecting the change of expression levels of the regulatory factors before and after differentiation of porcine muscle stem cells, and thus judges the differentiation condition of cultured pork.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set for detecting porcine muscle regulatory factors by real-time fluorescence quantitative PCR and an application thereof. Background technique [0002] At present, people are trying to use cell-based cultured meat instead of artificial breeding to obtain high-quality muscle tissue, which can improve production efficiency and reduce environmental impact. Compared with traditionally produced meat, its production is convenient and fast, and it improves food safety. The generation of skeletal muscle is a continuous and complex process, mainly the process of proliferation and differentiation of stem cells. Muscle stem cells (MuSC) are currently the main stem cells used in cultivating meat. [0003] The muscle regulatory factors (muscleregulatoryfactors, MRFs) family, the MRFs family genes are only specifically expressed in skeletal muscle cell lines, and are necessary for the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2600/158C12Q2600/124C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 王守伟李雨爽李石磊李莹莹
Owner CHINA MEAT RES CENT
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