CN111455055A - Human TYMS gene expression level detection standard reference substance - Google Patents
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Abstract
The invention provides a human TYMS gene expression quantity detection standard reference substance, which is prepared by taking cDNA reverse transcribed from RNA extracted from a blood standard substance as a template, adding a specific primer and a PCR reaction reagent for detecting TYMS gene and β -actin gene expression levels, carrying out PCR amplification, comparing an amplification result with a standard curve obtained by taking human TYMS gene with gradient concentration and β -actin gene detection standard substance as templates to obtain TYMS gene expression quantity, and mixing the standard substances in equal volume to obtain a detection standard reference substance.
Description
Technical Field
The invention is applied to the technical field of gene detection, and particularly relates to a human TYMS gene expression level detection standard reference substance.
Background
The fluorine drugs comprise 5-FU, tegafur, carmofur, floxuridine, doxifluridine, capecitabine and the like, have good curative effects on digestive tract tumors and other solid tumors, and play an important role in medical treatment of tumors. After entering human body, the fluorine-containing medicine can be converted into pyrimidine nucleotide analogues such as active metabolite fluorouracil monophosphate deoxynucleoside (FdUMP), fluorouracil triphosphate deoxynucleoside (FdUTP) and fluorouracil triphosphate (FUTP), and further generates antitumor activity.
Thymidylate Synthase (TS) encoded by TYMS (Thymidylate synthase) gene is a rate-limiting enzyme for pyrimidine nucleotide synthesis, is an important factor for tumor growth, and is also a target enzyme for fluorine drugs to exert cytotoxic effect. After the fluorine-containing drug is converted into FdUMP, the FdUMP is combined with TS and 5, 10-methylene tetrahydrofolate to form a triple compound, and after the dUMP is combined with the TS, the dUMP is converted into deoxythymidine dTMP, so that the DNA synthesis is limited. Clinical studies of various tumors have shown that the expression level of TYMS mRNA is closely related to the therapeutic effect of fluorine drugs, for example: colorectal cancer, lung cancer, breast cancer, head and neck squamous cell carcinoma, and the like. Clinical studies show that tumor patients with low TYMS mRNA level have good effect of receiving fluorine drug chemotherapy, and the median survival time is long. On the contrary, patients with high TYMS expression have poor curative effect on fluorine treatment.
At present, in a clinical tumor marker detection method, traditional technologies such as an immunohistochemistry method (chemical staining), a flow cytometry method and an immunoradiometric method are mainly applied, but the method has the defects of excessively complicated experimental process, large subjective influence on result reading and the like. In recent years, the fluorescence quantitative PCR gradually replaces the traditional detection method in clinic by virtue of the advantages of short detection time, high detection sensitivity, capability of real-time detection, initial content of a reaction gene in a tissue, short detection time, avoidance of occurrence of remaining pollution and the like. Common methods for fluorescent quantitative PCR include SYBR Green I dye method and TaqMan probe method. Among them, SYBRGreen I is not as specific as TaqMan because it is a non-saturation dye, and must be judged by observing a dissolution curve.
A commonly used detection method for detecting the TYMS gene expression quantity in the existing literature and report is to artificially synthesize a standard substance on the basis of fluorescent quantitative PCR, and then judge the TYMS gene expression quantity by a relative quantitative method. The method is mainly used for judging the expression level of the TYMS of a sample to be detected clinically based on the median of a statistical sample, and only the expression quantity of the TYMS gene in the sample relative to an artificially synthesized standard can be detected by taking the artificially synthesized standard as a reference, but the median of the TYMS mRNA level relative large data in the sample cannot be truly reflected. Therefore, the significance of the single detection of the expression quantity of the TYMS mRNA in clinical medication guidance is limited, and the clinical diagnosis still needs large-scale statistical samples to judge the expression level of the TYMS mRNA in a certain sample. Therefore, the significance of separately detecting the copy number of the TYMS mRNA in clinical medication guidance is limited, and the clinical level of the expression level of the TYMS mRNA in a certain sample can be judged only by large-scale statistics of the sample. If large-scale statistical sample analysis is required for clinical medication guidance in each test, the whole test process is complicated and the cost is high.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a human TYMS gene expression level detection standard reference substance. The standard reference substance can be used for evaluating the level of TYMS mRNA in a sample relative to the median, and is simple, convenient and quick.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a preparation method of a human TYMS gene expression level detection standard reference substance comprises the following steps:
1) taking cDNA reverse transcribed by RNA extracted from a blood standard substance as a template, adding a specific primer and a PCR reaction reagent for detecting the TYMS gene expression level, carrying out PCR amplification, and obtaining the TYMS gene expression quantity (initial copy number) N by contrasting an amplification result with a TYMS gene standard curve obtained by taking a human TYMS gene detection standard substance with gradient concentration as a template1/μl；
2) Taking cDNA reverse transcribed by RNA extracted from blood standard as a template, adding specific primers and PCR reaction reagents for β -actin gene expression level detection, carrying out PCR amplification, and obtaining β -actin gene expression amount (initial copy number) N by contrasting an amplification result with a β -actin gene standard curve obtained by taking human β -actin gene detection standard with gradient concentration as a template2/μl；
3) Diluting the human TYMS gene detection standard substance and the human β -actin gene detection standard substance, and mixing the diluted substances in equal volumes to obtain a standard reference substance.
Further, the preparation method of the TYMS Gene detection standard in the step 1) comprises the steps of constructing a plasmid according to a TYMS Gene (Gene ID:7298) sequence published in an NCBI database, synthesizing a Gene fragment of the sequence, inserting the fragment into a T vector, transforming by using an Escherichia coli DH5 α strain, and extracting the plasmid to serve as the TYMS Gene detection standard.
Further, the base sequence (SEQ ID NO:7) of the TYMS gene detection standard is:
CCAGGTGACTTTATACACACTTTGGGAGATGCACATATTTACCTGAATCACATCGAGCCACTGAAAATTCAGCTTCAGCGAGAACCCAGACCTTTCCCAAAGCTCAGGATTCTTCGAAAAGTTGAGAAAATTGATGACTTCAAAGCTGAAGACTTTCAGATTGAAGGGTACAATCCGCATCCAACTATTAAAATGGAAATGGCTGTTTAG。
further, the preparation method of the β -actin Gene detection standard in the step 2) comprises the steps of constructing a plasmid according to a β -actin Gene (Gene ID:60) sequence published by NCBI database, synthesizing a Gene fragment of the sequence, inserting the fragment into a T vector, transforming by using an Escherichia coli DH5 α strain, and extracting the plasmid as the β -actin Gene detection standard.
Further, the base sequence (SEQ ID NO:8) of the β -actin gene detection standard is as follows:
ACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAG。
further, the conditions of PCR amplification in the steps 1) and 2) are as follows:
the first stage is as follows: 2 minutes at 95 ℃ for 1 cycle;
and a second stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 5 cycles;
and a third stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃ (signal collected), 30 seconds at 72 ℃, 40 cycles;
signal collection: the third stage collects the VIC signal at 60 ℃ and performs real-time PCR.
Further, a preparation method of a human TYMS gene expression level detection standard reference substance, which comprises the following steps:
1) taking a human TYMS gene detection standard product with gradient concentration as a template, and adding a specific primer, a probe and a PCR reaction reagent for detecting the TYMS gene expression level to form a PCR reaction system I; carrying out PCR amplification on the PCR reaction system I, and drawing a TYMS gene standard curve;
2) adding cDNA reverse transcribed by RNA extracted from a blood standard product into the PCR reaction system I as a template; carrying out PCR amplification on the PCR reaction system I, comparing the amplification result with the TYMS gene standard curve in the step 1), and obtaining the TYMS gene expression quantity (initial copy number) N1/μl；
3) Using human β -actin gene detection standard with gradient concentration as a template, adding specific primers, probes and PCR reaction reagents for detecting β -actin gene expression level to form a PCR reaction system II, carrying out PCR amplification on the PCR reaction system II, and drawing a β -actin gene standard curve;
4) adding cDNA reverse transcribed by RNA extracted from a blood standard substance into the PCR reaction system II as a template, carrying out PCR amplification on the PCR reaction system II, comparing the amplification result with the β -actin gene standard curve in the step 3), and obtaining β -actin gene expression quantity (initial copy number) N2/μl；
5) Respectively diluting the human TYMS gene detection standard substance and the human β -actin gene detection standard substance to 2 × N1/μl、2×N1After mul, the standard reference substance is obtained after equal volume mixing.
A human TYMS gene expression quantity detection standard reference substance obtained by the preparation method is used for detecting TYMS gene expression level and β -actin gene expression level, and the nucleotide sequence of the specific primer and the probe is as follows:
TYMS gene upstream primer, TYMS-F: CACATATTTACCTGAATCACATC (SEQ ID NO:1),
downstream primer of TYMS gene, TYMS-R: TAATAGTTGGATGCGGATTGTA (SEQ ID NO:2),
TYMS gene detection probe, TYMS-P: FAM-TTCCCTCTGCTGACAACCAAACGT-TRAMA (SEQ ID NO: 3);
β -actin gene upstream primer, β -actin-F: CCGACAGGATGCAGAAGGAG (SEQ ID NO:4),
β -actin gene downstream primer, β -actin-R: AGGATGGAGCCGCCGAT (SEQ ID NO:5),
β -actin gene detecting probe, β -actin-P: VIC-CACCCAGCACAATGAAGATCAAGATCA-TRAMA (SEQ ID NO: 6).
Further, a fluorescent group at the 5 'end of the probe for detecting the TYMS gene expression level is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, the fluorescent reporter group is FAM, VIC, HEX, cy5 or ROX, a quenching group at the 3' end is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR, and the fluorescent quenching group is TAMRA, BHQ1, BHQ2, MGB or Dabcy 1.
Further, the fluorescence reporter group at the 5 'end is FAM, and the fluorescence quencher group at the 3' end is TAMRA.
Further, a 5 'end fluorescent group of the probe for detecting the expression level of β -actin gene is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, the fluorescent reporter group is FAM, VIC, HEX, cy5 or ROX, a 3' end quenching group is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR, and the fluorescent quenching group is TAMRA, BHQ1, BHQ2, MGB or Dabcy 1.
Further, the fluorescence reporter group at the 5 'end is VIC, and the fluorescence quencher group at the 3' end is TAMRA.
Compared with the prior art, the invention has the beneficial effects that:
(1) taking the median of TYMS gene and β -actin gene expression of a patient as standard reference, artificially constructing a simulation median of TYMS gene and β -actin gene standard substances, and preparing a standard reference substance which can be stored for a long time;
(2) the method judges the level of the TYMS gene expression level through the standard reference substance, does not need to perform large-scale sample statistics to determine the clinical median value, is simple to operate, and saves time and cost.
Drawings
FIG. 1 is a TYMS gene standard curve of a human TYMS gene expression level detection standard control product of the present invention.
FIG. 2 is a β -actin gene standard curve of a human TYMS gene expression level detection standard control of the present invention.
FIG. 3 is a standard reference substance amplification curve of the human TYMS gene expression level detection standard reference substance of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
In addition, unless otherwise specifically indicated, various starting materials, reagents, instruments and equipment used in the present invention may be commercially available or prepared by existing methods.
Example 1: composition of human TYMS gene expression quantity detection standard reference substance
1. Primer group and probe for detecting TYMS gene expression level detection:
TYMS gene upstream primer, TYMS-F: CACATATTTACCTGAATCACATC (SEQ ID NO:1)
Downstream primer of TYMS gene, TYMS-R: TAATAGTTGGATGCGGATTGTA (SEQ ID NO:2)
TYMS gene detection probe, TYMS-P: FAM-TTCCCTCTGCTGACAACCAAACGT-TRAMA (SEQ ID NO:3)
The 5 'end fluorescent group of the probe is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, FAM, VIC, HEX, cy5 or ROX is selected, the 3' end quenching group is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR, and TAMRA, BHQ1, BHQ2, MGB or Dabcy1 is selected. In a preferred embodiment, the fluorescent group at the 5 'end of the detection probe is FAM and the fluorescence quenching group at the 3' end of the detection probe is TAMRA.
2. Primer group and probe for detecting expression level of reference gene β -actin:
β -actin gene upstream primer, β -actin-F: CCGACAGGATGCAGAAGGAG (SEQ ID NO:4)
β -actin gene downstream primer, β -actin-R: AGGATGGAGCCGCCGAT (SEQ ID NO:5)
β -actin gene detecting probe, β -actin-P: VIC-CACCCAGCACAATGAAGATCAAGATCA-TRAMA (SEQ ID NO:6)
The 5 'end fluorescent group of the probe is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, FAM, VIC, HEX, cy5 or ROX is selected, the 3' end quenching group is a conventionally used fluorescent quenching group suitable for fluorescent quantitative PCR, and TAMRA, BHQ1, BHQ2, MGB or Dabcy1 is selected. In a preferred embodiment, the fluorescent group at the 5 'end of the detection probe is VIC, and the fluorescence quenching group at the 3' end of the detection probe is TAMRA.
3. Blood standard product
1001 patients were collected and their blood samples were tested for the expression level of TYMS gene and β -actin gene in the blood of 1001 patients using primer set and probe for detecting the expression level of TYMS gene in 1 and 2 and primer set and probe for detecting the expression level of β -actin gene in 1001 patients, and cDNA reverse transcribed from RNA extracted from blood samples of 1001 median patients (TYMS gene expressed in β -actin gene) was selected as blood standards.
4. TYMS gene detection standard substance
According to the sequence of TYMS Gene (Gene ID:7298) published by NCBI database, making plasmid construction by using Gene engineering method, synthesizing Gene fragment of said sequence, inserting said fragment into T vector, utilizing Escherichia coli DH5 α strain to make transformation and extracting plasmid, using nanodrop 2000 to measure concentration and purity for stand-by use, and using said fragment as TYMS Gene detection standard product, the base sequence of TYMS Gene detection standard product is as follows:
CCAGGTGACTTTATACACACTTTGGGAGATGCACATATTTACCTGAATCACATCGAGCCACTGAAAATTCAGCTTCAGCGAGAACCCAGACCTTTCCCAAAGCTCAGGATTCTTCGAAAAGTTGAGAAAATTGATGACTTCAAAGCTGAAGACTTTCAGATTGAAGGGTACAATCCGCATCCAACTATTAAAATGGAAATGGCTGTTTAG(SEQ ID NO:7)
diluting TYMS gene detection standard with known copy number to 1 × 10 times of gradient7、1×106、1×105、1×104、1×103copy/u L for use.
5.β -actin gene detection standard substance
According to the β -actin Gene (Gene ID:60) sequence published by NCBI database, constructing plasmid by Gene engineering method, synthesizing the Gene fragment of the sequence, inserting the fragment into T vector, transforming and extracting plasmid by using Escherichia coli DH5 α strain, measuring concentration and purity of nanodrop 2000 for later use as β -actin Gene detection standard substance, β -actin Gene detection standard substance base sequence is as follows:
ACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAG(SEQ ID NO:8)
diluting β -actin gene detection standard with known copy number to 1 × 10 times of gradient dilution7、1×106、1×105、1×104、1×103copy/u L for use.
6. PCR reaction solution
The PCR reaction solution contains the primers and probes described in 1 and 2, and also contains hot-start taq enzyme, buffer, magnesium ions, dNTPs and the like required for the PCR reaction.
Example 2: preparation method of human TYMS gene expression quantity detection standard reference substance
A preparation method of a human TYMS gene expression level detection standard reference substance comprises the following steps:
1) taking a human TYMS gene detection standard product with gradient concentration as a template, and adding a specific primer, a probe and a PCR reaction reagent for detecting the TYMS gene expression level to form a PCR reaction system I; and performing PCR amplification on the PCR reaction system I, and drawing a TYMS gene standard curve.
Referring to the attached figure 1, collecting a fresh blood sample of 1001 patients, preparing a TYMS gene detection standard according to the method of 4 in example 1, adding the specific primers and the probe for detecting the TYMS gene expression level in example 1, and adding a PCR reaction reagent to form a PCR reaction system I; and performing PCR amplification on the PCR reaction system I, and drawing a TYMS gene standard curve. The PCR reaction system I is as follows:
2) adding cDNA reverse transcribed by RNA extracted from a blood standard product into the PCR reaction system I in the step 1) as a template; carrying out PCR amplification on the PCR reaction system I, comparing the amplification result with the TYMS gene standard curve in the step 1), and obtaining the TYMS gene expression quantity (initial copy number) N1/μl。
The PCR amplification conditions were:
the first stage is as follows: 2 min at 95 ℃ for 1 cycle
And a second stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 5 cycles;
and a third stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃ (signal collected), 30 seconds at 72 ℃, 40 cycles;
signal collection: the third stage collects FAM and VIC signals at 60 ℃ and performs real-time PCR.
3) Referring to the attached figure 2, β -actin gene detection standard is prepared by the method of 5 in example 1, human β -actin gene detection standard with gradient concentration is used as a template, specific primers, probes and PCR reaction reagents for detecting β -actin gene expression level are added to form a PCR reaction system II, the PCR reaction system II is subjected to PCR amplification, and a β -actin gene standard curve is drawn, wherein the PCR reaction system II comprises the following components:
| component name | Volume (μ l) |
| 5×colorless Goq Flexi Buffer | 4.0 |
| Mgcl2 solution(25mM) | 2.0 |
| PCR nucleotide(10mM) | 0.5 |
| Hot start polymerase(5U) | 0.15 |
| β-actin-F(10uM) | 0.5 |
| β-actin-R(10uM) | 0.5 |
| β-actin-P(10uM) | 0.4 |
| Template | 1.0 |
| DDH2O | Make up to 20.0 |
4) Adding cDNA reverse transcribed by RNA extracted from a blood standard substance into the PCR reaction system II in the step 3) as a template, carrying out PCR amplification on the PCR reaction system II, and contrasting the amplification result with the β -actin gene standard curve in the step 3) to obtain β -actin geneExpression amount (initial copy number) N2/μl。
The PCR amplification conditions were:
the first stage is as follows: 2 min at 95 ℃ for 1 cycle
And a second stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 5 cycles;
and a third stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃ (signal collected), 30 seconds at 72 ℃, 40 cycles;
signal collection: the third stage, collecting FAM and VIC signals at 60 ℃, performing real-time PCR, and recording the Δ Ct, which is Ct for each sample(TYMS)-Ct(β-actin)。
5) Diluting the human TYMS gene detection standard to 2 × N1Mu.l human β -actin gene detection standard diluted to 2 × N2And after mul, mixing the two diluted gene detection standard substances in equal volume uniformly to obtain a standard reference substance.
Example 3: detection of human TYMS gene expression level detection standard reference substance
In order to better verify the effect of the standard, a standard reference substance verification experiment is performed in the second embodiment, and the verification experiment includes the following steps:
the cDNA of the median patient obtained in 3 of example 1 and the standard control prepared in example 2 were used as templates for quantitative fluorescence PCR.
The PCR system composition is as follows:
| component name | Volume (μ l) |
| 5×colorless |
4 |
| Mgcl2 solution(25mM) | 2 |
| PCR nucleotide(10mM) | 0.5 |
| Hot start polymerase(5U) | 0.15 |
| TYMS-F(10uM) | 0.5 |
| TYMS-R(10uM) | 0.5 |
| TYMS-P(10uM) | 0.4 |
| β-actin-F(10uM) | 0.5 |
| β-actin-R(10uM) | 0.5 |
| β-actin-P(10uM) | 0.4 |
| |
1 |
| DDH2O | Make up to 20.0 |
The PCR amplification conditions were:
the first stage is as follows: 2 min at 95 ℃ for 1 cycle
And a second stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 5 cycles;
and a third stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 40 cycles;
signal collection: the third stage collects FAM and VIC signals at 60 ℃ and performs real-time PCR.
The experimental results are as follows:
referring to the attached figure 3 of the specification, the cDNA of the median patient sample is basically consistent with the detection amplification curve of the relative expression quantity of β -actin gene in the TYMS gene in the prepared standard reference substance.
Therefore, the standard reference substance prepared in the invention can directly evaluate the expression level of a certain sample TYMS gene, does not need to detect the sample in a large scale to determine a median value, can compare the expression level of the TYMS gene in the detected sample and directly guide clinical medication, and is convenient and rapid.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. It will be understood by those skilled in the art that various changes, substitutions of equivalents, and alterations can be made without departing from the spirit and scope of the invention.
Sequence listing
<110> Chongqing Puluotong Gene medical research institute Co., Ltd
<120> a human TYMS gene expression level detection standard reference substance
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ccaggtgact ttatacacac tttgggagat gcacatattt acctgaatca catcgagcca 60
ctgaaaattc agcttcagcg agaacccaga cctttcccaa agctcaggat tcttcgaaaa 120
gttgagaaaa ttgatgactt caaagctgaa gactttcaga ttgaagggta caatccgcat 180
ccaactatta aaatggaaat ggctgtttag 210
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ctcctgagcg caagtactcc gtgtggatcg gcggctccat cctggcctcg ctgtccacct 180
tccagcagat gtggatcagc aagcaggag 209
Claims (8)
1. A preparation method of a human TYMS gene expression level detection standard reference substance is characterized by comprising the following steps:
1) taking cDNA reverse transcribed by RNA extracted from a blood standard substance as a template, adding a specific primer and a PCR reaction reagent for detecting the TYMS gene expression level, carrying out PCR amplification, and contrasting an amplification result with a TYMS gene standard curve obtained by taking a human TYMS gene detection standard substance with gradient concentration as a template to obtain the TYMS gene expression quantity (initial copy number) N1/mul;
2) taking cDNA reverse transcribed by RNA extracted from a blood standard product as a template, adding a specific primer and a PCR reaction reagent for β -actin gene expression level detection, carrying out PCR amplification, and comparing an amplification result with a β -actin gene standard curve obtained by taking a human β -actin gene detection standard product with gradient concentration as a template to obtain β -actin gene expression quantity (initial copy number) N2/mul;
3) diluting the human TYMS gene detection standard substance and the human β -actin gene detection standard substance, and mixing the diluted substances in equal volumes to obtain a standard reference substance.
2. The method for preparing a standard control for detecting the expression level of the human TYMS Gene according to claim 1, wherein the TYMS Gene detection standard in the step 1) is prepared by constructing a plasmid based on a TYMS Gene (Gene ID:7298) sequence published in NCBI database, synthesizing a Gene fragment of the sequence, inserting the fragment into a T-vector, transforming with Escherichia coli DH5 α strain, and extracting the plasmid as the TYMS Gene detection standard.
3. The method for preparing the standard control for detecting the expression level of the human TYMS Gene according to claim 1, wherein the β -actin Gene detection standard in the step 2) is prepared by constructing a plasmid based on a β -actin Gene (Gene ID:60) sequence published in NCBI database, synthesizing a Gene fragment of the sequence, inserting the Gene fragment into a T vector, transforming with Escherichia coli DH5 α strain, and extracting the plasmid as the β -actin Gene detection standard.
4. The method for preparing the standard reference substance for detecting the expression level of the human TYMS gene in claim 1, wherein the method comprises the following steps: the PCR amplification conditions in the steps 1) and 2) are as follows:
the first stage is as follows: 2 minutes at 95 ℃ for 1 cycle;
and a second stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ and 5 cycles;
and a third stage: 30 seconds at 95 ℃, 30 seconds at 60 ℃ (signal collected), 30 seconds at 72 ℃, 40 cycles;
signal collection: the third stage collects the VIC signal at 60 ℃ and performs real-time PCR.
5. The method for preparing the standard reference substance for detecting the expression level of the human TYMS gene according to any one of claims 1 to 4, wherein the standard reference substance comprises: a preparation method of a human TYMS gene expression level detection standard reference substance comprises the following steps:
1) taking a human TYMS gene detection standard product with gradient concentration as a template, and adding a specific primer, a probe and a PCR reaction reagent for detecting the TYMS gene expression level to form a PCR reaction system I; carrying out PCR amplification on the PCR reaction system I, and drawing a TYMS gene standard curve;
2) reverse transcription of RNA extracted from blood standardscDNA is taken as a template, and the cDNA is added into the PCR reaction system I; carrying out PCR amplification on the PCR reaction system I, comparing the amplification result with the TYMS gene standard curve in the step 1), and obtaining the TYMS gene expression quantity (initial copy number) N1/μl；
3) Using human β -actin gene detection standard with gradient concentration as a template, adding specific primers, probes and PCR reaction reagents for detecting β -actin gene expression level to form a PCR reaction system II, carrying out PCR amplification on the PCR reaction system II, and drawing a β -actin gene standard curve;
4) adding cDNA reverse transcribed by RNA extracted from a blood standard substance into the PCR reaction system II as a template, carrying out PCR amplification on the PCR reaction system II, comparing the amplification result with the β -actin gene standard curve in the step 3), and obtaining β -actin gene expression quantity (initial copy number) N2/μl；
5) Respectively diluting the human TYMS gene detection standard substance and the human β -actin gene detection standard substance to 2 × N1/μl、2×N2After mul, the standard reference substance is obtained after equal volume mixing.
6. A human TYMS gene expression level detection standard control obtained by the preparation method of any one of claims 1 to 5, which is characterized in that the specific primers and probe nucleotide sequences used for TYMS gene expression level detection and β -actin gene expression level detection are as follows:
TYMS gene upstream primer, TYMS-F: CACATATTTACCTGAATCACATC the flow of the air in the air conditioner,
downstream primer of TYMS gene, TYMS-R: TAATAGTTGGATGCGGATTGTA the flow of the air in the air conditioner,
TYMS gene detection probe, TYMS-P: FAM-TTCCCTCTGCTGACAACCAAACGT-TRAMA;
β -actin gene upstream primer, β -actin-F: CCGACAGGATGCAGAAGGAG,
β -actin gene downstream primer, β -actin-R: AGGATGGAGCCGCCGAT,
β -actin gene detecting probe, β -actin-P, VIC-CACCCAGCACAATGAAGATCAAGATCA-TRAMA.
7. The human TYMS gene expression level detection standard control substance of claim 6, which is characterized in that: the fluorescent group at the 5 'end of the probe for detecting the TYMS gene expression level is a conventionally used fluorescent reporter group suitable for fluorescent quantitative PCR analysis, the fluorescent reporter group is FAM, VIC, HEX, cy5 or ROX, the quencher group at the 3' end is a conventionally used fluorescent quencher group suitable for fluorescent quantitative PCR, and the fluorescent quencher group is TAMRA, BHQ1, BHQ2, MGB or Dabcy 1.
8. The human TYMS gene expression level detection standard control substance of claim 6, wherein the 5 '-end fluorescent group of the probe for detecting the expression level of β -actin gene is a conventionally used fluorescent reporter group suitable for quantitative fluorescent PCR analysis, the fluorescent reporter group is FAM, VIC, HEX, cy5 or ROX, the 3' -end quencher group is a conventionally used fluorescent quencher group suitable for quantitative fluorescent PCR, and the fluorescent quencher group is TAMRA, BHQ1, BHQ2, MGB or Dabcy 1.
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Effective date of registration: 20220428 Address after: 400700 Xinmao Road, Beibei District, Beibei District, Chongqing (free trade area) Patentee after: Chongqing puluotong Life Technology Group Co.,Ltd. Address before: 400711 9-1, building 3, 128 Anli Road, Beiwenquan street, Beibei District, Chongqing Patentee before: Chongqing puluotong Gene Medicine Research Institute Co.,Ltd. |
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Denomination of invention: A standard reference for the detection of human TYMS gene expression Effective date of registration: 20220727 Granted publication date: 20211116 Pledgee: Chongqing Rural Commercial Bank Co.,Ltd. Beibei sub branch Pledgor: Chongqing puluotong Life Technology Group Co.,Ltd. Registration number: Y2022500000048 |
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Address after: No. 117-896 Yunhan Avenue, Beibei District, Chongqing, 400700 Patentee after: Chongqing puluotong Life Technology Group Co.,Ltd. Country or region after: China Address before: 400700 Xinmao Road, Beibei District, Beibei District, Chongqing (free trade area) Patentee before: Chongqing puluotong Life Technology Group Co.,Ltd. Country or region before: China |