CN111455055B - Human TYMS gene expression level detection standard reference substance - Google Patents

Human TYMS gene expression level detection standard reference substance Download PDF

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CN111455055B
CN111455055B CN202010350372.2A CN202010350372A CN111455055B CN 111455055 B CN111455055 B CN 111455055B CN 202010350372 A CN202010350372 A CN 202010350372A CN 111455055 B CN111455055 B CN 111455055B
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韩勋领
孙松松
罗锋
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Chongqing Puluotong Life Technology Group Co ltd
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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 beta-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 beta-actin gene detection standard substance as templates, obtaining TYMS gene expression quantity, and mixing the standard substances in equal volumes to obtain a detection standard reference substance. The invention utilizes the median of the artificially constructed gene standard substance simulation population sample as a control for detection, and uses hot-start enzyme to ensure the accuracy of the detection result and the stability of the system.

Description

Human TYMS gene expression level detection standard reference substance
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, SYBR Green I is not as specific as TaqMan method because it is a non-saturation dye, and its specificity 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 a blood standard substance as a template, adding a specific primer and a PCR reaction reagent for detecting the expression level of the beta-actin gene, carrying out PCR amplification, and obtaining the expression quantity (initial copy number) N of the beta-actin gene by comparing the amplification result with a beta-actin gene standard curve obtained by taking a human beta-actin gene detection standard substance with gradient concentration as a template2/μl；
3) Diluting the human TYMS gene detection standard substance and the human beta-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 substance in the step 1) comprises the following steps: constructing plasmids according to a TYMS Gene (Gene ID:7298) sequence published by NCBI database; the sequence gene fragment is synthesized, then the fragment is inserted into a T vector, and a plasmid is transformed and extracted by using an Escherichia coli DH5 alpha strain to be used as a TYMS gene detection standard product.
Further, the base sequence (SEQ ID NO:7) of the TYMS gene detection standard is:
CCAGGTGACTTTATACACACTTTGGGAGATGCACATATTTACCTGAATCACATCGAGCCACTGAAAATTCAGCTTCAGCGAGAACCCAGACCTTTCCCAAAGCTCAGGATTCTTCGAAAAGTTGAGAAAATTGATGACTTCAAAGCTGAAGACTTTCAGATTGAAGGGTACAATCCGCATCCAACTATTAAAATGGAAATGGCTGTTTAG。
further, the preparation method of the beta-actin gene detection standard in the step 2) comprises the following steps: constructing plasmids according to a beta-actin Gene (Gene ID:60) sequence published by an NCBI database; synthesizing the sequence gene fragment, then inserting the fragment into a T vector, transforming by using an escherichia coli DH5 alpha strain and extracting a plasmid as a beta-actin gene detection standard substance.
Further, the base sequence (SEQ ID NO:8) of the beta-actin gene detection standard substance 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) Taking a human beta-actin 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 expression level of the beta-actin gene to form a PCR reaction system II; carrying out PCR amplification on the PCR reaction system II, and drawing a beta-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 beta-actin gene standard curve in the step 3), and obtaining the beta-actin gene expression quantity (initial copy number) N2/μl；
5) Respectively diluting the human TYMS gene detection standard substance and the human beta-actin gene detection standard substance to 2 XN1/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 beta-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);
beta-actin gene upstream primer, beta-actin-F: CCGACAGGATGCAGAAGGAG (SEQ ID NO:4),
beta-actin gene downstream primer, beta-actin-R: AGGATGGAGCCGCCGAT (SEQ ID NO:5),
beta-actin gene detection probe, beta-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 the beta-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 beta-actin gene expression of a patient as standard reference, artificially constructing a simulation median of TYMS gene and beta-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 beta-actin gene standard curve of a human TYMS gene expression level detection standard reference substance 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. The primer group and the probe for detecting the expression level of the reference gene beta-actin are as follows:
beta-actin gene upstream primer, beta-actin-F: CCGACAGGATGCAGAAGGAG (SEQ ID NO:4)
Beta-actin gene downstream primer, beta-actin-R: AGGATGGAGCCGCCGAT (SEQ ID NO:5)
Beta-actin gene detection probe, beta-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 1001 patients using 1 and 2 primer sets and probes for detecting the expression level of TYMS gene and β -actin gene in 1001 patients, and cDNA reverse transcribed from RNA extracted from 1001 patients (TYMS gene expressed in β -actin gene) blood samples was selected as blood standards.
4. TYMS gene detection standard substance
Constructing plasmid by genetic engineering method according to the sequence of TYMS Gene (Gene ID:7298) published by NCBI database; synthesizing the sequence gene fragment, then inserting the fragment into a T vector, transforming and extracting a plasmid by using an Escherichia coli DH5 alpha strain, and measuring the concentration and the purity of nanodrop 2000 for later use, wherein the concentration and the purity are used as TYMS gene detection standards. The base sequence of the TYMS gene detection standard substance is as follows:
CCAGGTGACTTTATACACACTTTGGGAGATGCACATATTTACCTGAATCACATCGAGCCACTGAAAATTCAGCTTCAGCGAGAACCCAGACCTTTCCCAAAGCTCAGGATTCTTCGAAAAGTTGAGAAAATTGATGACTTCAAAGCTGAAGACTTTCAGATTGAAGGGTACAATCCGCATCCAACTATTAAAATGGAAATGGCTGTTTAG(SEQ ID NO:7)
diluting TYMS gene detection standard with known copy number to 1 × 10 gradient7、1×106、1×105、1×104、1×103copy/uL for use.
5. Beta-actin gene detection standard substance
Constructing plasmids by a genetic engineering method according to a beta-actin Gene (Gene ID:60) sequence published by an NCBI database; synthesizing the sequence gene fragment, then inserting the fragment into a T vector, transforming and extracting a plasmid by using an escherichia coli DH5 alpha strain, and measuring the concentration and purity of nanodrop 2000 for later use, wherein the concentration and purity are used as a beta-actin gene detection standard product. The base sequence of the beta-actin gene detection standard substance is as follows:
ACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAG(SEQ ID NO:8)
diluting the beta-actin gene detection standard substance with known copy number to 1 × 10 by 10-fold gradient7、1×106、1×105、1×104、1×103copy/uL 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:
Figure BDA0002471728000000061
Figure BDA0002471728000000071
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, a beta-actin gene detection standard is prepared by the method of 5 in the embodiment 1, and a PCR reaction system II is formed by taking a human beta-actin gene detection standard with gradient concentration as a template and adding a specific primer, a probe and a PCR reaction reagent for detecting the expression level of the beta-actin gene; and performing PCR amplification on the PCR reaction system II, and drawing a beta-actin gene standard curve. The PCR reaction system II is as follows:
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 beta-actin gene standard curve in the step 3) to obtain the beta-actin gene expression quantity (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 above human TYMS gene detection standard to 2 XN1Mu.l, human beta-actin gene detection standard dilution is 2 XN2And 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 Goq Flexi Buffer 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
Template 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 beta-actin gene expression quantity of 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
<160> 8
<170> SIPOSequenceListing 1.0
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<400> 1
cacatattta cctgaatcac atc 23
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
taatagttgg atgcggattg ta 22
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttccctctgc tgacaaccaa acgt 24
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgacaggat gcagaaggag 20
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aggatggagc cgccgat 17
<210> 6
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cacccagcac aatgaagatc aagatca 27
<210> 7
<211> 210
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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
<210> 8
<211> 209
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acctgtacgc caacacagtg ctgtctggcg gcaccaccat gtaccctggc attgccgaca 60
ggatgcagaa ggagatcact gccctggcac ccagcacaat gaagatcaag atcattgctc 120
ctcctgagcg caagtactcc gtgtggatcg gcggctccat cctggcctcg ctgtccacct 180
tccagcagat gtggatcagc aagcaggag 209

Claims (6)

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, comparing 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, and obtaining the TYMS gene expression quantity N by counting the initial copy number1/μl；
2) 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 expression level of the beta-actin gene to carry out PCR amplification, comparing an amplification result with a beta-actin gene standard curve obtained by taking a human beta-actin gene detection standard substance with gradient concentration as a template, and obtaining the expression quantity N of the beta-actin gene by counting the initial copy number2/μl；
3) Respectively diluting the human TYMS gene detection standard substance and the human beta-actin gene detection standard substance to 2 XN1 /μl、2×N 2After mul, uniformly mixing the solution in equal volume to obtain a standard reference substance;
the base sequence of the TYMS gene detection standard substance is shown in SEQ ID NO. 7; the base sequence of the beta-actin gene detection standard substance is shown as SEQ ID NO. 8;
collecting 1001 patient blood samples, detecting TYMS gene and beta-actin gene expression levels in 1001 patient blood by using a primer group and a probe for detecting TYMS gene expression levels and a primer group and a probe for detecting beta-actin gene expression levels, and selecting 1001 median patient blood samples as blood standard products according to the TYMS gene expression levels relative to the beta-actin gene expression levels;
wherein, the primer group and the probe for detecting the TYMS gene expression level are as follows:
TYMS gene upstream primer, TYMS-F: CACATATTTACCTGAATCACATC
Downstream primer of TYMS gene, TYMS-R: TAATAGTTGGATGCGGATTGTA
TYMS gene detection probe, TYMS-P: FAM-TTCCCTCTGCTGACAACCAAACGT-TRAMA
Beta-actin gene upstream primer, beta-actin-F: CCGACAGGATGCAGAAGGAG
Beta-actin gene downstream primer, beta-actin-R: AGGATGGAGCCGCCGAT
Beta-actin gene detection probe, beta-actin-P: VIC-CACCCAGCACAATGAAGATCAAGATCA-TRAMA.
2. 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 preparation method of the TYMS gene detection standard substance in the step 1) comprises the following steps: constructing plasmids according to a TYMS Gene sequence which is published by an NCBI database and has the serial number of Gene ID: 7298; the sequence gene fragment is synthesized, then the fragment is inserted into a T vector, and a plasmid is transformed and extracted by using an Escherichia coli DH5 alpha strain to be used as a TYMS gene detection standard product.
3. 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 preparation method of the beta-actin gene detection standard substance in the step 2) comprises the following steps: constructing plasmids according to a beta-actin Gene sequence which is published by an NCBI database and has the serial number of Gene ID: 60; synthesizing the sequence gene fragment, then inserting the fragment into a T vector, transforming by using an escherichia coli DH5 alpha strain and extracting a plasmid as a beta-actin gene detection standard substance.
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 ℃, 30 seconds at 72 ℃ and 40 cycles;
signal collection: the third stage collects FAM and VIC signals 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: the method 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 N by counting the initial copy number1 /μl；
3) Taking a human beta-actin 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 expression level of the beta-actin gene to form a PCR reaction system II; carrying out PCR amplification on the PCR reaction system II, and drawing a beta-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 beta-actin gene standard curve in the step 3), and obtaining the beta-actin gene expression quantity N by counting according to the initial copy number2 /μl；
5) Respectively diluting the human TYMS gene detection standard substance and the human beta-actin gene detection standard substance to 2 XN1 /μl、2×N 2After mul, the standard reference substance is obtained after equal volume mixing.
6. A human TYMS gene expression level detection standard control obtained by the production method according to any one of claims 1 to 5.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012125828A2 (en) * 2011-03-15 2012-09-20 The University Of North Carolina At Chapell Hill Methods of treating breast cancer with anthracycline therapy
CN103088122A (en) * 2012-12-07 2013-05-08 周宏灏 Kit and method for detecting expression level of TYMS (Thymidylate Synthetase) mRNA (messenger Ribonucleic Acid)
CN103602672A (en) * 2013-11-04 2014-02-26 北京海思特临床检验所有限公司 Primer, probe and kit for detection of gene expression amounts and use methods thereof
CN103757094A (en) * 2013-11-15 2014-04-30 陕西佰美基因股份有限公司 Fluorescent PCR based method for detecting polymorphism of TYMS gene
CN105255865A (en) * 2015-10-16 2016-01-20 苏州贝斯麦医疗仪器有限公司 Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit
CN107760786A (en) * 2017-11-29 2018-03-06 广东大臻医学检验所有限公司 Primer sets, kit and method for the detection of tumor chemotherapeutic drug related gene TYMS expressions
CN110964818A (en) * 2019-11-28 2020-04-07 重庆浦洛通基因医学研究院有限公司 Detection kit and detection method for human BRAF gene V600E mutation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2512153B (en) * 2008-11-17 2014-11-12 Veracyte Inc Methods and compositions of molecular profiling for disease diagnostics
CN102453748A (en) * 2010-10-18 2012-05-16 北京雅康博生物科技有限公司 Plasmid standard substance for quantitative detection by fluorescence quantitative PCR
US20140141986A1 (en) * 2011-02-22 2014-05-22 David Spetzler Circulating biomarkers
CA2877378A1 (en) * 2012-06-29 2014-01-03 Nanostring Technologies, Inc. Methods of treating breast cancer with gemcitabine therapy

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012125828A2 (en) * 2011-03-15 2012-09-20 The University Of North Carolina At Chapell Hill Methods of treating breast cancer with anthracycline therapy
CN103088122A (en) * 2012-12-07 2013-05-08 周宏灏 Kit and method for detecting expression level of TYMS (Thymidylate Synthetase) mRNA (messenger Ribonucleic Acid)
CN103602672A (en) * 2013-11-04 2014-02-26 北京海思特临床检验所有限公司 Primer, probe and kit for detection of gene expression amounts and use methods thereof
CN103757094A (en) * 2013-11-15 2014-04-30 陕西佰美基因股份有限公司 Fluorescent PCR based method for detecting polymorphism of TYMS gene
CN105255865A (en) * 2015-10-16 2016-01-20 苏州贝斯麦医疗仪器有限公司 Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit
CN107760786A (en) * 2017-11-29 2018-03-06 广东大臻医学检验所有限公司 Primer sets, kit and method for the detection of tumor chemotherapeutic drug related gene TYMS expressions
CN110964818A (en) * 2019-11-28 2020-04-07 重庆浦洛通基因医学研究院有限公司 Detection kit and detection method for human BRAF gene V600E mutation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Microarray analysis reveals down-regulation of the tumour suppressor gene WWOX and up-regulation of the oncogene TYMS in intracranial sporadic meningiomas;Mads Aarhus等;《J Neurooncol》;20080326;第88卷;第251-259页 *
晚期肺腺癌组织中TYMS基因表达水平判定及其临床意义验证;张志豪等;《武警医学》;20150115(第01期);第32-35、38页 *
构建荧光定量PCR法检测高发区肝癌TYMS基因及临床意义;厦门市第三医院;《中国科技成果数据库》;20150101;第1-2页 *

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