CN111826451A - Primer group for real-time fluorescent quantitative PCR detection of porcine muscle regulatory factor and application thereof - Google Patents
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Abstract
The invention discloses a primer group for detecting porcine muscle regulatory factor by real-time fluorescence quantitative PCR and application thereof, belonging to the technical field of biology. The invention provides a specific primer pair and an internal reference primer pair capable of detecting the pig muscle regulating factors MyoD and MyoG, cDNA is obtained by extracting sample RNA and carrying out reverse transcription, real-time fluorescence PCR is carried out by utilizing the provided primers, the mRNA expression quantity of the pig muscle regulating factors MyoD and MyoG can be rapidly, specifically, sensitively and stably detected, and the method can be used for detecting the change of the expression level of the regulating factors before and after the differentiation of the pig muscle stem cells, thereby judging the differentiation condition of the pig cultivated meat.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting a porcine muscle regulatory factor by real-time fluorescent quantitative PCR and application thereof.
Background
At present, people try to obtain high-quality muscle tissues by replacing artificial feeding with cell culture-based meat, and the mode can improve the production efficiency and reduce the influence on the environment. Compared with the meat produced by the traditional method, the production is convenient and fast, and the food safety is improved. Skeletal muscle formation is a continuous and complex process, mainly the process of stem cell proliferation and differentiation. Muscle stem cells (MuSC) are the stem cells mainly used for culturing meat at present.
The family of Myogenesis Regulators (MRFs), MRFs family genes, are specifically expressed only in skeletal muscle cell lines and are essential in skeletal muscle cell determination and differentiation, including four transcription factors, MyoD, MyoG, Myf5 and Myf 6. The MyoD and Myf5 genes control the differentiation of somite cells into myoblasts, the differentiation of myoblasts into mature skeletal muscle cells is controlled by MyoG, and the Myf6 gene is expressed at the late stage of muscle cell differentiation or after muscle cell differentiation. Myoblasts were established at the early MyoD gene, followed by MyoG gene mediated terminal differentiation of myoblasts.
At present, the research on meat cultivation is still in the initial stage, and various methods for detecting the expression of the myogenesis regulatory factor are still in short supply. With the rapid development of molecular biology technology and its wide application in medical research, it has become possible to detect gene expression by Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time PCR) technology. However, no report is available for determining the differentiation degree of the cultured meat by detecting the expression of muscle regulatory factors in the cultured meat by real-time fluorescence quantitative reverse transcription polymerase chain reaction.
Disclosure of Invention
The invention aims to provide a primer group for detecting a porcine muscle regulatory factor by real-time fluorescence quantitative PCR and application thereof, so as to solve the problems in the prior art, conveniently detect the expression of the porcine muscle regulatory factor in cultured meat, and simply and quickly judge the differentiation degree of the cultured meat.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a primer group for detecting the expression quantity of porcine muscle regulatory factors, which comprises primer pairs I and II of two regulatory factors of MyoD and MyoG and an internal reference primer pair III, wherein the sequences of the primer pairs are shown as follows;
MyoD-F:5'-CACTACAGCGGTGACTCAGA-3'；
MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3'；
(2) specific primer pair for detecting MyoG:
MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3'；
MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3'；
(3) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
the invention also provides a primer group for detecting the expression quantity of the pork muscle regulatory factor, the primer group comprises a primer pair for detecting the regulatory factor MyoD and an internal reference primer pair, and the sequences of the primer pairs are as follows:
(1) specific primer pairs for detecting MyoD:
MyoD-F:5'-CACTACAGCGGTGACTCAGA-3'；
MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3'；
(2) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
the invention also provides a primer group for detecting the expression quantity of the pork muscle regulatory factor, which is characterized by comprising a primer pair for detecting the regulatory factor MyoG and an internal reference primer pair, wherein the sequences of the primer pairs are as follows:
(1) specific primer pair for detecting MyoG:
MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3'；
MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3'；
(2) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
the invention also provides a kit for detecting the expression level of the porcine muscle regulatory factor, and the kit comprises any primer group described above.
Preferably, the kit further comprises DNTPS, Mg2+, DNA polymerase, reverse transcriptase, RNase inhibitor and SYBR Green I.
The invention also provides a detection method for detecting the expression level of the muscle regulatory factor in the sample, which comprises the following steps:
(1) extracting total RNA from the sample to be detected, and carrying out reverse transcription reaction to obtain cDNA to be detected;
(2) taking the cDNA to be detected as a template, and carrying out real-time fluorescence quantitative PCR reaction on a Roche480 fluorescence quantitative PCR instrument by using any primer group or any kit described above;
(3) and after the reaction is finished, calculating the expression quantity of the muscle regulatory factor in the sample to be detected according to the self-contained software of the Roche480 fluorescent quantitative PCR instrument.
Preferably, the primers used in step (2) are the same in amount and each final concentration is 10. mu. mol/L, and the real-time fluorescent quantitative PCR reaction is performed at 95 ℃ for 5 minutes, 95 ℃ for 30 seconds and 60 ℃ for 30 seconds for 40 cycles of amplification.
The invention also provides application of the primer pair or the kit or the detection method in detecting the proliferation and differentiation degree of the muscle stem cells in the pork cultivated by the pig.
Preferably, the degree of proliferation and differentiation of the sample is determined by detecting the relative expression levels of MyoD and MyoG in the sample with respect to the first day of culture.
Preferably, when the expression level of MyoD and MyoG is increased relative to that of the muscle stem cell before proliferation and differentiation, the cell is indicated to start differentiation, and if the relative expression level of MyoG is higher than that of MyoD, the cell is indicated to have differentiated into a muscle cell.
The invention has the following technical effects:
the invention provides a primer group, a kit and a detection method for detecting the expression quantity of a pig muscle regulation factor, which can specifically and simultaneously detect the expression quantities of the pig muscle regulation factors MyoD and MyoG, and have high sensitivity and accuracy and convenient operation. The invention also provides an application of the primer group, the kit and the detection method in detecting the proliferation and differentiation degree of the muscle stem cells in the pork cultivated meat, and the proliferation and differentiation conditions of the cultivated meat can be simply and conveniently judged by detecting and calculating the relative expression quantity of MyoD and MyoG relative to the beginning of the cultivation, so that a foundation is laid for the subsequent artificial regulation and control of the growth and differentiation of the cultivated meat.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the expression trends of MyoD and MyoG in pig muscle stem cells at 1, 2, 5 and 6 days;
FIG. 2 is a graph showing the expression trend of MyoG in porcine muscle stem cells at 1, 3 and 5 days;
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The "parts" in the present invention are all parts by mass unless otherwise specified.
Example 1 fluorescent quantitative RT-PCR method for simultaneously detecting MyoD and MyoG expression before and after muscle cell differentiation
Firstly, materials:
RNA extraction kits were purchased from Qiagen. Reverse transcription kits and the like were purchased from Takara. TBGreenPremix Ex TaqII is a product of Takara.
Secondly, primer and probe design and synthesis:
the primer is designed by taking the MyoD full-length cDNA sequence (Gen Bank accession number 407604) as a template, and the optimal primer is selected from the MyoD full-length cDNA sequence by considering the MyoD genome DNA sequence condition.
The sequence of the PCR upstream primer for detection is as follows:
5’-CACTACAGCGGTGACTCAGA-3'(SEQ ID NO.1),
the sequence of the downstream primer is as follows:
5’-GCTGTAATAGGTGCCGTCGT-3'(SEQ ID NO.2),
the primer is designed by taking the MyoG full-length cDNA sequence (Gen Bank accession number 497618) as a template, and the optimal primer is selected from the MyoG full-length cDNA sequence by considering the MyoG genome DNA sequence condition.
The sequence of the upstream primer is as follows:
5’-CTTCTACCAGGAACCCCACTTCT-3'(SEQ IDNO.3)，
the sequence of the downstream primer is as follows:
5’-GTCCCCAGCCCCTTATCTTC-3'(SEQ ID NO.4)，
the sequence information of the internal reference primer pair is as follows:
GAPDH-F:5′-ACTCACTCTTCTACCTTTGATGCT-3'(SEQ IDNO.5)，
GAPDH-R:5′-TGTTGCTGTAGCCAAATTCA-3'(SEQ IDNO.6)，
the above primers were synthesized by thermo, and the primers were stored at-20 ℃ to minimize repeated freeze-thawing.
Third, the detection of the standard article
Qiagen RNeasy Mini kit, tip used and EP tube were all dedicated to RNA extraction.
1. Extraction of sample RNA
Culturing muscle stem cells, taking three holes of 6 pore plates of cells cultured for 1 day and 2 days, changing a differentiation culture medium for continuous culture on the third day of culture, taking 3 holes of 6 pore plates of cells cultured for 5 days and 6 days: without digestion, the culture broth was thoroughly blotted dry and then lysed by repeated pipetting with the recommended amount of lysate RLT Plus (see Table 1). Qiagen RNeasy Mini kit for RNA extraction.
TABLE 1 amounts of lysis buffer RLT Plus
| Culture vessel | Number of cells | Lysate RLTPlus |
| ＜6cm | ＜5×106 | 350ul |
| 6-10cm | ≤1×107 | 600ul |
2. Reverse transcription of RNA into cDNA
Total volume 20ul, divided into the following two steps, all operating on ice:
(1) removal of genomic DNA
The reaction mixture was prepared on ice according to tables 2 and 3, and in order to ensure the accuracy of the reaction mixture preparation, Master Mix was prepared in the amount of reaction number + 2, and then dispensed into each reaction tube, and finally RNA sample was added.
TABLE 2 genomic DNA removal reaction mixture
Mix gently and incubate at 42 ℃ for 2 minutes, or 5 minutes at room temperature.
(2) Reverse transcription reaction
TABLE 3 reverse transcription reaction System
| Name of reagent | Volume of |
| Reaction solution of |
10.0μl |
| Prime Script RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0 |
| 5×PrimeScriptBuffer 2(for Real Time) | 4.0μl |
| RNase-free ddH2O | 4.0μl |
| Total of | 20μl |
Mix gently, incubate at 37 ℃ for 15 minutes, heat at 85 ℃ for 5 seconds, and inactivate the enzyme.
Performing reverse transcription on the mixed solution in a total reaction volume of 20u1, detecting, performing PCR amplification on a Roche480 fluorescent quantitative PCR instrument by using upstream and downstream primers, preparing a PCR reaction premixed solution on ice according to a system shown in the table 4, taking suction errors into consideration, wherein the volume of the prepared premixed solution is at least more than 10% of the total volume of all reaction solutions, performing PCR for 5 minutes at 95 ℃, performing 40 cycles of amplification at 30 seconds at 95 ℃ and 30 seconds at 60 ℃, and each group is provided with three repeat holes. Simultaneously, an internal reference primer is added for correction. The MyoD and MyoG expression levels of the test specimen are calculated from the measurement results.
TABLE 4 PCR System
| Name of reagent | Volume of |
| SYBR(2×) | 5μl |
| Forward primer (10. mu.M) | 0.2μl |
| Reverse primer (10. mu.M) | 0.2μl |
| cDNA | 0.5μl |
| H2O | 4.1μl |
Fourth, the detection result of the sample
The experiment is repeated for three times, and the expression trends of MyoD and MyoG of the pig muscle stem cells at 1, 2, 5 and 6 days are shown in a figure 2: MyoD and MyoG participate in the differentiation and specialization process of myoblasts, myoblasts are established by MyoD genes at an early stage, and then the MyoG genes mediate terminal differentiation of the myoblasts. Therefore, the MyoD and MyoG expression levels are gradually increased in the proliferation culture of the pork stem cells, and the MyoD is higher than the MyoG in the early stage. When the stem cell gradually differentiated into a muscle cell, the expression amount of MyoD began to gradually decrease, and the expression amount of MyoG was higher than MyoD.
Example 2 verification of MyoD detection by fluorescent quantitative RT-PCR
Porcine muscle stem cells cultured for 1 day and 2 days were selected, and experiments were performed according to the method of example 1 using only the primers shown in SEQ ID NO.1 and SEQ ID NO.2 and the internal reference primer, and the results are shown in Table 5.
Table 5 results of the reproducibility test of the MyoD assay:
the Ct value of MYoD is increased and has good reproducibility compared to internal controls.
Example 3 fluorescent quantitative RT-PCR method for detecting MyoG expression
Porcine muscle stem cells cultured for 1 day, 3 days and 5 days were selected, and experiments were performed according to the method of example 1 using only the primers shown in SEQ ID NO.3 and SEQ ID NO.4 and the internal reference primer, and the results are shown in FIG. 2 and Table 6.
TABLE 6 repeatability test results for MyoG assay
The Ct value of MYoG is increased and has good reproducibility compared to internal controls.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> China meat food comprehensive research center
Primer group for detecting porcine muscle regulatory factor by real-time fluorescent quantitative PCR (polymerase chain reaction) and application thereof
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gctgtaatag gtgccgtcgt 20
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gtccccagcc ccttatcttc 20
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Claims (10)
1. The primer group for detecting the expression quantity of the pork muscle regulatory factor is characterized by comprising a primer pair of two regulatory factors of MyoD and MyoG and an internal reference primer pair, wherein the sequences of the primer pairs are shown as follows;
(1) specific primer pairs for detecting MyoD:
MyoD-F:5'-CACTACAGCGGTGACTCAGA-3'；
MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3'；
(2) specific primer pair for detecting MyoG:
MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3'；
MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3'；
(3) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
2. the primer group for detecting the expression quantity of the pork muscle regulatory factor is characterized by comprising a primer pair for detecting the regulatory factor MyoD and an internal reference primer pair, wherein the sequences of the primer pairs are as follows:
(1) specific primer pairs for detecting MyoD:
MyoD-F:5'-CACTACAGCGGTGACTCAGA-3'；
MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3'；
(2) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
3. the primer group for detecting the expression quantity of the pork muscle regulatory factor is characterized by comprising a primer pair for detecting the regulatory factor MyoG and an internal reference primer pair, wherein the sequences of the primer pairs are as follows:
(1) specific primer pair for detecting MyoG:
MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3'；
MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3'；
(2) internal reference primers:
GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'；
GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'。
4. a kit for detecting the expression level of a pork muscle regulatory factor, which is characterized by comprising the primer group of any one of claims 1 to 3.
5. The kit of claim 4, further comprising DNTPS, Mg2+, DNA polymerase, reverse transcriptase, RNase inhibitor and SYBR Green I.
6. A method for detecting the expression level of a muscle regulatory factor in a sample, comprising the steps of:
(1) extracting total RNA from the sample to be detected, and carrying out reverse transcription reaction to obtain cDNA to be detected;
(2) performing real-time fluorescent quantitative PCR reaction on a Roche480 fluorescent quantitative PCR instrument by using the cDNA to be detected as a template and using the primer group of any one of claims 1 to 3 or the kit of any one of claims 4 to 5;
(3) and after the reaction is finished, calculating the expression quantity of the muscle regulatory factor in the sample to be detected according to the self-contained software of the Roche480 fluorescent quantitative PCR instrument.
7. The detection method according to claim 6, wherein the primers used in step (2) are used in the same amount and the final concentration is 10 μmol/L, and the real-time fluorescent quantitative PCR reaction is performed by 40 cycles of amplification at 95 ℃ for 5 minutes, 95 ℃ for 30 seconds and 60 ℃ for 30 seconds.
8. Use of the primer set according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 or the detection method according to any one of claims 6 to 7 for detecting the proliferation and differentiation degree of muscle stem cells in pork-meat.
9. The use of claim 8, wherein the relative expression levels of MyoD and MyoG in the sample are measured relative to the first day of culture to determine the degree of proliferation and differentiation of the sample.
10. The use of claim 9, wherein an increase in the expression of MyoD, MyoG relative to that of myostem cells prior to proliferation and differentiation indicates that the cell has begun to differentiate, and wherein an increase in the relative expression of MyoG relative to MyoD indicates that the cell has differentiated into a muscle cell.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016168890A1 (en) * | 2015-04-22 | 2016-10-27 | Genea Ip Holdings Pty Ltd | Generation of muscle-lineage cells from stem cells |
| CN108753779A (en) * | 2018-06-07 | 2018-11-06 | 天津农学院 | Ox lncRNA-133a and the application in the regulation and control of bovine muscle satellite cell Proliferation, Differentiation and verification method |
| CN110564743A (en) * | 2019-09-10 | 2019-12-13 | 宁夏农林科学院固原分院 | CircR-UQCC1 gene of Lianshan cattle, over-expression vector, construction method and application thereof |
-
2020
- 2020-09-11 CN CN202010955451.6A patent/CN111826451A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016168890A1 (en) * | 2015-04-22 | 2016-10-27 | Genea Ip Holdings Pty Ltd | Generation of muscle-lineage cells from stem cells |
| CN108753779A (en) * | 2018-06-07 | 2018-11-06 | 天津农学院 | Ox lncRNA-133a and the application in the regulation and control of bovine muscle satellite cell Proliferation, Differentiation and verification method |
| CN110564743A (en) * | 2019-09-10 | 2019-12-13 | 宁夏农林科学院固原分院 | CircR-UQCC1 gene of Lianshan cattle, over-expression vector, construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| E.A. LEE等: "Effects of variation in porcine MYOD1 gene on muscle fiber characteristics, lean meat production, and meat quality traits", 《MEAT SCIENCE》 * |
| MARINUS F. W. TE PAS等: "Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family", 《MOLECULAR BIOLOGY REPORTS》 * |
| 王珊等: "牛MyoG基因启动子的克隆与序列分析", 《西北农林科技大学学报（自然科学版）》 * |
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