Primer set for detecting porcine myostatin by real-time fluorescent quantitative PCR and application thereof

Abstract

The application discloses a primer set for real-time fluorescent quantitative PCR detection of a pig muscle regulatory factor and application thereof, and belongs to the technical field of biology. The application provides specific primer pairs capable of detecting pig muscle regulatory factors MyoD and MyoG and reference primer pairs. cDNA is obtained by extracting sample RNA and performing reverse transcription, and real-time fluorescent PCR is performed by using the provided primers, so that the mRNA expression amount of the pig muscle regulatory factors MyoD and MyoG can be simultaneously detected quickly, specifically, sensitively and stably, and the application can be used for detecting the change in the expression level of the regulatory factors before and after the differentiation of pig muscle stem cells, so that the differentiation condition of pig breeding meat can be judged.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to primer sets for real-time quantitative PCR detection of porcine muscle regulatory factors and their applications. Background Technology

[0002] Currently, researchers are exploring cell-cultured meat as an alternative to artificially raised meat to obtain high-quality muscle tissue. This method can improve production efficiency and reduce environmental impact. Compared to traditionally produced meat, it is easier and faster to produce and improves food safety. Skeletal muscle formation is a continuous and complex process, primarily involving the proliferation and differentiation of stem cells. Muscle stem cells (MuSCs) are currently the main stem cell type used in cultured meat.

[0003] The muscle regulatory factors (MRFs) family consists of genes specifically expressed only in skeletal muscle cell lines. These genes are essential for the determination and differentiation of skeletal muscle cells and include four transcription factors: MyoD, MyoG, Myf5, and Myf6. MyoD and Myf5 genes control the differentiation of somatic cells into myoblasts, while MyoG regulates the differentiation of myoblasts into mature skeletal muscle cells. Myf6 is expressed late in or after myoblast differentiation. Early on, the MyoD gene establishes myoblasts, and subsequently, the MyoG gene mediates terminal differentiation of myoblasts.

[0004] Research on cultured meat is still in its early stages, and methods for detecting the expression of myogenic regulatory factors are lacking. With the rapid development of molecular biology techniques and their widespread application in medical research, detecting gene expression using real-time quantitative reverse transcription polymerase chain reaction (Real-time PCR) has become possible. However, there are currently no reports on using Real-time quantitative reverse transcription polymerase chain reaction to detect the expression of muscle regulatory factors in cultured meat to determine its differentiation degree. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set for real-time quantitative PCR detection of porcine muscle regulatory factors and its application, so as to solve the problems existing in the prior art, thereby facilitating the detection of the expression of muscle regulatory factors in cultured meat and quickly and easily determining the degree of differentiation of cultured meat.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a primer set for detecting the expression levels of regulatory factors in porcine muscle. The primer set includes primer pairs I and II for two regulatory factors, MyoD and MyoG, and an internal reference primer pair III. The sequences of the primer pairs are shown below.

[0008] MyoD-F:5'-CACTACAGCGGTGACTCAGA-3';

[0009] MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3';

[0010] (2) Specific primer pairs for detecting MyoG:

[0011] MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3';

[0012] MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3';

[0013] (3) Internal reference primer:

[0014] GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3';

[0015] GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'.

[0016] This invention also provides a primer set for detecting the expression level of a regulatory factor in pork muscle. The primer set includes a primer pair for detecting the regulatory factor MyoD and an internal reference primer pair. The sequences of the primer pairs are shown below:

[0017] (1) Specific primer pairs for detecting MyoD:

[0018] MyoD-F:5'-CACTACAGCGGTGACTCAGA-3';

[0019] MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3';

[0020] (2) Internal reference primer:

[0021] GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3';

[0022] GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'.

[0023] This invention also provides a primer set for detecting the expression level of a regulatory factor in pork muscle, characterized in that the primer set includes a primer pair for detecting the regulatory factor MyoG and an internal reference primer pair, the sequences of which are shown below:

[0024] (1) Specific primer pairs for detecting MyoG:

[0025] MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3';

[0026] MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3';

[0027] (2) Internal reference primer:

[0028] GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3';

[0029] GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'.

[0030] The present invention also provides a kit for detecting the expression levels of regulatory factors in porcine muscle, the kit comprising any of the primer sets described above.

[0031] Preferably, the kit also includes DNTPS, Mg2+, DNA polymerase, reverse transcriptase, RNase inhibitor and SYBR Green I.

[0032] This invention also provides a method for detecting the expression level of muscle regulatory factors in a sample, comprising the following steps:

[0033] (1) Total RNA was extracted from the sample to be tested and reverse transcription was performed to obtain cDNA to be tested;

[0034] (2) Using the cDNA to be tested as a template, perform real-time quantitative PCR reaction on a Roche 480 fluorescence quantitative PCR instrument using any of the primer sets or any of the kits described above.

[0035] (3) After the reaction was completed, the expression level of muscle regulatory factor in the sample was calculated according to the software of the Roche 480 real-time PCR instrument.

[0036] Preferably, the amount of primers used in step (2) is the same, and the final concentration is 10 μmol / L. The program of the real-time fluorescence quantitative PCR reaction is 95℃ for 5 minutes, 95℃ for 30 seconds, and 60℃ for 30 seconds, for 40 cycles of amplification.

[0037] The present invention also provides the application of the primer pairs, kits, or detection methods described herein in detecting the degree of proliferation and differentiation of muscle stem cells in cultured pork.

[0038] 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 relative to the first day of culture.

[0039] Preferably, when the expression levels of MyoD and MyoG begin to increase relative to the pre-differentiation stage of muscle stem cells, it indicates that the cells have begun to differentiate. If the relative expression level of MyoG is higher than that of MyoD, it indicates that the cells have differentiated into muscle cells.

[0040] The present invention has the following technical effects:

[0041] This invention provides a primer set, kit, and detection method for detecting the expression levels of porcine muscle regulatory factors MyoD and MyoG. These methods can specifically and simultaneously detect the expression levels of MyoD and MyoG, exhibiting high sensitivity and accuracy, and are easy to operate. This invention also provides the application of the primer set, kit, and detection method in detecting the degree of proliferation and differentiation of muscle stem cells in cultured pork. By detecting and calculating the relative expression levels of MyoD and MyoG compared to the beginning of culture, the proliferation and differentiation status of cultured meat can be easily determined, laying the foundation for subsequent artificial regulation of the growth and differentiation of cultured meat. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 The expression trends of MyoD and MyoG in porcine muscle stem cells on days 1, 2, 5, and 6;

[0044] Figure 2 The expression trend of MyoG in porcine muscle stem cells at 1, 3, and 5 days. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0051] Example 1: Simultaneous detection of MyoD and MyoG expression before and after muscle cell differentiation using real-time quantitative RT-PCR.

[0052] I. Materials:

[0053] RNA extraction kit was purchased from Qiagen. Reverse transcription kit and other equipment were purchased from Takara. TBGreenPremix Ex TaqII is a product of Takara.

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

[0055] Primers were designed using the full-length cDNA sequence of MyoD (Gen Bank accession number 407604) as a template, while also taking into account the MyoD genomic DNA sequence, and the best primers were selected from them.

[0056] The upstream primer sequence for PCR detection is:

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

[0058] The downstream primer sequence is:

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

[0060] Primers were designed using the full-length MyoG cDNA sequence (Gen Bank accession number 497618) as a template, while also taking into account the MyoG genomic DNA sequence, and the best primers were selected from them.

[0061] The upstream primer sequence is:

[0062] 5'-CTTCTACCAGGAACCCCACTTCT-3'(SEQ IDNO.3),

[0063] The downstream primer sequence is:

[0064] 5'-GTCCCCAGCCCCTTATCTTC-3'(SEQ ID NO.4),

[0065] The sequence information of the internal reference primer pair is as follows:

[0066] GAPDH-F:5′-ACTCACTCTTCTACCTTTGATGCT-3′(SEQ IDNO.5),

[0067] GAPDH-R:5′-TGTTGCTGTAGCCAAATTCA-3′(SEQ IDNO.6),

[0068] All primers were synthesized by Thermo Fisher Scientific and stored at -20°C to minimize repeated freeze-thaw cycles.

[0069] III. Standard Product Testing

[0070] The Qiagen RNeasy Mini kit uses pipette tips and EP tubes specifically designed for RNA extraction.

[0071] 1. RNA extraction from the sample

[0072] Muscle stem cells were cultured, and cells cultured for 1 day and 2 days were collected from 3 wells of a 6-well plate. On the third day of culture, the culture medium was changed and the cells were cultured for another 3 wells of a 6-well plate on the 5th and 6th days. No digestion was required. After thoroughly aspirating the culture medium, the recommended amount of lysis buffer RLT Plus (see Table 1) was added directly, and the cells were repeatedly pipetted to lyse. RNA was extracted using the Qiagen RNeasy Mini kit.

[0073] Table 1 Quantity of RLT Plus lysate

[0074] Petri dishes Cell count lysis buffer RLTPlus <6cm <![CDATA[<5×10 6 ]]> 350ul 6-10cm <![CDATA[≤1×10 7 ]]> 600ul

[0075] 2. RNA is reverse transcribed into cDNA

[0076] The total volume is 20ul, and the procedure is divided into the following two steps, all of which are performed on ice:

[0077] (1) Removal of genomic DNA

[0078] Prepare the reaction mixtures on ice according to Tables 2 and 3. To ensure the accuracy of the reaction mixture preparation, prepare the Master Mix by the reaction number + 2 before each reaction, then dispense it into each reaction tube, and finally add the RNA sample.

[0079] Table 2 Genomic DNA Removal Reaction Mixture

[0080]

[0081]

[0082] Mix gently and incubate at 42 degrees Celsius for 2 minutes, or at room temperature for 5 minutes.

[0083] (2) Reverse transcription reaction

[0084] Table 3 Reverse Transcription Reaction System

[0085] Reagent Name volume The reaction solution in step 1 10.0μl Prime Script RT Enzyme Mix I 1.0μl RT Primer Mix 1.0μl 5×PrimeScriptBuffer 2(for Real Time) 4.0μl <![CDATA[RNase-free ddH2O]]> 4.0μl total 20μl

[0086] Mix gently, incubate at 37 degrees Celsius for 15 minutes, and heat at 85 degrees Celsius for 5 seconds to inactivate the enzyme.

[0087] After reverse transcription in a total reaction volume of 20 μL, PCR amplification was performed on a Roche 480 real-time PCR instrument using upstream and downstream primers. The PCR reaction premix was prepared on ice according to the system described in Table 4. Considering aspiration error, the volume of the premix should be at least 10% larger than the total volume of all reactions. PCR conditions were 95℃ for 5 minutes denaturation, followed by 40 cycles of amplification at 95℃ for 30 seconds and 60℃ for 30 seconds, with three replicate wells per group. Internal control primers were added for correction. The expression levels of MyoD and MyoG in the samples were calculated from the results.

[0088] Table 4 PCR System

[0089] Reagent Name volume SYBR(2×) 5μl Forward primer (10 μM) 0.2μl Reverse primer (10 μM) 0.2μl cDNA 0.5μl <![CDATA[H2O]]> 4.1μl

[0090] IV. Sample Test Results

[0091] The expression trends of MyoD and MyoG in porcine muscle stem cells on days 1, 2, 5, and 6 were observed in three repeated experiments. Figure 2MyoD and MyoG are involved in the differentiation and specialization of myoblasts. In the early stages, the MyoD gene establishes myoblasts, followed by the MyoG gene mediating their terminal differentiation. Therefore, during the proliferation and culture of porcine stem cells, the expression levels of both MyoD and MyoG gradually increase, with MyoD initially showing a higher expression level than MyoG. As stem cells gradually differentiate into muscle cells, the expression level of MyoD begins to gradually decrease, while the expression level of MyoG remains higher than that of MyoD.

[0092] Example 2: Validation of MyoD detection by real-time RT-PCR

[0093] Porcine muscle stem cells cultured for 1 day and 2 days were selected, and experiments were conducted using only the primers and internal control primers shown in SEQ ID NO. 1 and SEQ ID NO. 2, following the method in Example 1. The results are shown in Table 5.

[0094] Table 5. Results of repeatability tests for the MyoD method:

[0095]

[0096] Compared to the internal reference, MYoD has a larger Ct value and good reproducibility.

[0097] Example 3: Detection of MyoG expression by real-time quantitative RT-PCR

[0098] Porcine muscle stem cells cultured for 1, 3, and 5 days were selected. Following the method in Example 1, experiments were conducted using only the primers and internal control primers shown in SEQ ID NO. 3 and SEQ ID NO. 4. The results are as follows: Figure 2 As shown in Table 6.

[0099] Table 6. Results of repeatability tests using the MyoG method

[0100]

[0101] Compared to the internal reference, MYoG has a larger Ct value and good reproducibility.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention. sequence list <110> China Meat Food Comprehensive Research Center <120> Primer set and its application for real-time quantitative PCR detection of porcine muscle regulatory factors <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 cactacagcg gtgactcaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 gctgtaatag gtgccgtcgt 20 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 cttctaccag gaaccccact tct 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gtccccagcc ccttatcttc 20 <210> 5 <211> twenty four <212> DNA <213> Artificial Sequence <400> 5 actcactctt ctacctttga tgct 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tgttgctgta gccaaattca 20

Claims

1. The application of a primer set for detecting the expression level of a regulatory factor in pork muscle in detecting the degree of proliferation and differentiation of muscle stem cells in cultured pork, characterized in that, The primer set includes primer pairs for two regulatory factors, MyoD and MyoG, and an internal reference primer pair. The primer pair sequences are shown below: (1) Specific primer pairs for detecting MyoD: MyoD-F:5'-CACTACAGCGGTGACTCAGA-3'; MyoD-R:5'-GCTGTAATAGGTGCCGTCGT-3'; (2) Specific primer pairs for detecting MyoG: MyoG-F:5'-CTTCTACCAGGAACCCCACTTCT-3'; MyoG-R:5'-GTCCCCAGCCCCTTATCTTC-3'; (3) Internal reference primer: GAPDH-F:5'-ACTCACTCTTCTACCTTTGATGCT-3'; GAPDH-R:5'-TGTTGCTGTAGCCAAATTCA-3'; The steps for detecting the degree of proliferation and differentiation of muscle stem cells in cultured pork are as follows: (1) Total RNA was extracted from the sample to be tested and reverse transcription was performed to obtain cDNA to be tested. (2) Using the cDNA to be tested as a template, a real-time quantitative PCR reaction was performed on a Roche 480 fluorescence quantitative PCR instrument using the primer set described above. (3) After the reaction, the expression level of muscle regulatory factor in the sample to be tested was calculated according to the software of the Roche 480 real-time PCR instrument. (4) The degree of proliferation and differentiation of the sample was determined by detecting the relative expression levels of MyoD and MyoG in the sample relative to the first day of culture; When the expression levels of MyoD and MyoG begin to increase relative to the pre-differentiation stage of muscle stem cells, it indicates that the cells have begun to differentiate. If the relative expression level of MyoG is higher than that of MyoD, it indicates that the cells have differentiated into muscle cells.

2. The application as described in claim 1, characterized in that, The amount of primers used in step (2) is the same, and the final concentration is 10 μmol / L. The program of the real-time fluorescence quantitative PCR reaction is 95℃ for 5 minutes, 95℃ for 30 seconds, and 60℃ for 30 seconds, for 40 cycles of amplification.

Images