CN111394467A - Detection reagent and method for expression quantity of cancer treatment drug related gene and application - Google Patents
Detection reagent and method for expression quantity of cancer treatment drug related gene and application Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention relates to the technical field of biology, in particular to a detection reagent for cancer treatment drug-associated gene expression quantity, wherein the detection reagent is a fluorescent quantitative PCR reagent, and the detection reagent comprises at least one of a primer pair and a fluorescent probe for detecting ERCC1, a primer pair and a fluorescent probe for detecting BRCA1, a primer pair and a fluorescent probe for detecting RRM1, and a primer pair and a fluorescent probe for detecting TYMS. The scheme provided by the invention can simultaneously detect the expression quantity of at least one of ERCC1, BRCA1, RRM1 and TYMS in the tumor tissue, and can provide reference for formulating the personalized treatment scheme of the liver cancer patient.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection reagent and a method for the expression level of a cancer treatment drug-associated gene and application thereof.
Background
Primary liver cancer is one of the most common digestive system malignant tumors in clinic, and the mortality rate of primary liver cancer is second in China and third in the world. In China, the incidence of liver cancer is very high due to the high infection rate of hepatitis B virus, and according to statistics, the number of liver cancer patients in China accounts for about more than half of the world. In clinical treatment, the 5-year survival rate of liver cancer patients is only about 10%. The treatment of liver cancer is mainly surgical treatment, in recent years, the field of basic research and transformation research of liver cancer is rapidly advanced, and the screening, molecular typing and development of individualized targeted drugs of molecular markers greatly improve the drug treatment effect of liver cancer patients, prolong the life cycle of the liver cancer patients and improve the life quality of the liver cancer patients.
At present, the variety of chemotherapy drugs is more, and how to select the chemotherapy drug suitable for a patient avoids ineffective harmful chemotherapy, and the improvement of the pertinence of clinical tumor treatment is an urgent problem to be solved.
Disclosure of Invention
The invention provides a detection reagent and a method for the expression quantity of related genes of cancer treatment drugs and application thereof, which can be used for simultaneously detecting the expression quantity of related genes of a plurality of liver cancer treatment drugs in tumor tissues of cancer patients so as to facilitate the personalized drug selection of the cancer patients.
In a first aspect, a detection reagent for liver cancer treatment drug-associated gene expression level is provided, the detection reagent is a fluorescent quantitative PCR reagent, and the detection reagent includes at least one of the following:
a primer pair and a fluorescent probe for detecting ERCC1, a primer pair and a fluorescent probe for detecting BRCA1, a primer pair and a fluorescent probe for detecting RRM1, and a primer pair and a fluorescent probe for detecting TYMS.
In some embodiments, the primer pair for detecting ERCC1 consists of the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent probe for detecting ERCC1 is shown in SEQ ID NO. 3.
In some embodiments, the primer pair for detecting BRCA1 consists of the sequence shown in SEQ ID No.4 and the sequence shown in SEQ ID No.5, and the nucleotide sequence of the fluorescent probe for detecting BRCA1 is shown in SEQ ID No. 6.
In some embodiments, the primer pair for detecting RRM1 consists of the sequence shown in SEQ ID No. 7and the sequence shown in SEQ ID No.8, and the nucleotide sequence of the fluorescent probe for detecting RRM1 is shown in SEQ ID No. 9.
In some embodiments, the primer pair for detecting TYMS consists of the sequence shown in SEQ ID NO.10 and the sequence shown in SEQ ID NO.11, and the nucleotide sequence of the fluorescent probe for detecting TYMS is shown in SEQ ID NO. 12.
In some embodiments, the cancer is primary liver cancer.
In a second aspect, a kit for detecting the expression level of a gene associated with a cancer therapeutic drug is provided, which comprises the detection reagent of the first aspect.
In one embodiment, the kit further comprises reverse transcriptase and Taq enzyme.
In a third aspect, a method for detecting an expression level of a gene related to a cancer therapeutic drug is provided, which comprises: extracting mRNA of the tumor tissue; in the same PCR reaction tube, reverse transcription PCR is carried out by taking mRNA of tumor tissues as a template, and fluorescence quantitative PCR is carried out by taking a reverse transcription product as a template; wherein the primer pair of the reverse transcription PCR and the fluorescence quantitative PCR is at least one of
A primer pair for detecting ERCC1, a primer pair for detecting BRCA1, a primer pair for detecting RRM1 and a primer pair for detecting TYMS.
In a fourth aspect, there is provided a use of the detection reagent of the first aspect in the preparation of a product for screening humanized cancer treatment drugs.
In some embodiments, the cancer is primary liver cancer.
The scheme provided by the invention can simultaneously detect the expression quantity of at least one of ERCC1, BRCA1, RRM1 and TYMS in the tumor tissue, and can provide reference for formulating the personalized treatment scheme of the liver cancer patient.
Detailed Description
The technical solution of the present invention is further described in detail by the following examples.
A large amount of clinical data show that the expression level of target gene mRNA such as ERCC1, BRCA1, RRM1, TYMS and the like in tumor tissues can predict the response of patients to common chemotherapeutic drugs such as platinum, gemcitabine, fluorouracil and the like. ERCC1, BRCA1, RRM1, TYMS may also be collectively referred to as cancer treatment drug related genes.
Clinical studies have been demonstrated as follows.
Excision repair cross-complementing gene 1 (ERCC 1) is involved in the occurrence of platinum chemotherapy drug resistance, and the expression level of the Excision repair cross-complementing gene is in negative correlation with the curative effect and the life cycle of platinum chemotherapy of various cancers, namely, a patient with low expression level of ERCC1 gene is sensitive to platinum drugs, and conversely, a patient with high expression level of ERCC1 gene shows drug resistance.
The curative effect of the anti-microtubule drug is closely related to the mRNA expression level of susceptibility gene 1 (BRCA 1) in tumor tissues, namely, patients with high expression level of BRCA1 gene are sensitive to the anti-microtubule drug, and conversely, patients with low expression level show drug resistance.
Tumor patients with low Ribonucleotide reductase M1 (Riboside reductase M1, RRM1) mRNA expression level have relatively high sensitivity to gemcitabine, good drug efficacy and prolonged median survival, whereas patients with high RRM1 mRNA expression should avoid gemcitabine.
Tumor patients with low Thymidylate synthase (TYMS) mRNA expression levels receive fluorine chemotherapy with better efficacy and longer median survival. On the contrary, patients with high TYMS mRNA expression have poor therapeutic effect on fluorine.
By detecting the expression quantity (mRNA expression level) of the genes in the tumor tissues of cancer patients, an individual treatment scheme can be formulated for the patients, which is beneficial to selecting chemotherapeutic drugs suitable for the patients, avoids ineffective harmful chemotherapy and improves the pertinence of tumor clinical treatment.
The invention provides a method for detecting the expression quantity of a cancer treatment drug related gene based on a real-time fluorescence PCR technology. The real-time fluorescence PCR technology is based on the principle of Fluorescence Resonance Energy Transfer (FRET), and mainly utilizes thermostable DNA polymerase (Taq enzyme) to have 5 'to 3' direction exonuclease activity while having 5 'to 3' direction polymerase activity. The TaqMan fluorescent probe is respectively marked with a fluorescent reporter group and a quenching group at the 5 ' end and the 3 ' end, and the 3 ' end of the probe is phosphorylated to prevent the probe from being extended in the PCR amplification process. When the probe is kept intact, the quenching group is close to the fluorescent reporter group, and the former inhibits the fluorescent emission of the latter; once the probe is hydrolyzed, the fluorescent reporter group is separated from the quenching group, and the inhibition effect is released. The renaturation period probe is hybridized with template DNA, the Taq enzyme in the extension period moves along the DNA template along with the extension of the primer, when the Taq enzyme is moved to the position of the probe, the 5 'to 3' exonuclease activity of the Taq enzyme can degrade the fluorescent probe hybridized with the template DNA, so that a fluorescent reporter group and a quenching group are separated, the inhibition effect is relieved, the fluorescence is emitted, and the signal can be automatically collected by an instrument. According to the relation between the fluorescence signal and the amplification cycle number, the amplification instrument software system can automatically calculate to obtain a real-time amplification curve. When the cycle number (Ct value) that the fluorescence signal passes when reaching the set threshold value is the cycle number, the amplification signal value exceeds the background fluorescence value and is detected, the Ct value and the logarithm of the initial copy number of the template have a linear relation, and the more the initial copy number is, the smaller the Ct value is.
In the scheme provided by the invention, a standard curve is firstly made for a target gene (namely a cancer treatment drug associated gene) and a housekeeping gene in a sample, whether the amplification efficiencies of the target gene and the housekeeping gene are consistent or close to each other is determined through the standard curve, and the amplification efficiencies are optimized to be consistent.
In some embodiments, the invention provides that the housekeeping gene is GAPDH.
Next, the scheme provided by the embodiment of the present invention is specifically described in each embodiment.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise stated, the experimental METHODS, detection METHODS, AND preparation METHODS disclosed herein are based on conventional techniques IN the art, such as molecular biology, biochemistry, Chromatin structure AND analysis, analytical chemistry, cell culture, recombinant DNA techniques, AND related fields, which are well described IN the prior art, see, IN particular, Sambrook et al MO L ECU L AR C L ONING: A L ABORATORATOR MANUA 8652, Second edition, Cold Spring Harbor L analysis Press, 1989AND Third edition, 2001, Ausubel et al, CURRENT PROTOCO L S INMO L ECU L AR BIO L OGY, John Wiley & Sons, New York, 1987AND biological orders, the devices will be used, METHODS of DIDIDISY 2 IN L, Actic, Experimental & Sons, Sandwith et al, sample # Press, AND Press, sample # 1998, sample # Press, AND P, sample # Press et al, AND sample # Press et al.
Example 1
Primers and probes for detecting the expression levels of ERCC1, BRCA1, RRM1, TYMS and for detecting the housekeeping gene GAPDH, respectively, were designed. The designed primers are shown in Table 1.
TABLE 1
| Sequence numbering | Sequence name | Nucleotide sequence 5 '-3' |
| SEQ ID NO.1 | ERCC1-1 | TTCCTCAGCCTACGCTACCA |
| SEQ ID NO.2 | ERCC1-2 | CTGCTGGAGATCTTTCACATC |
| SEQ ID NO.3 | ERCC1-P | ACAAGCAGGACCTGCAAT |
| SEQ ID NO.4 | BRCA1-1 | CTGTCTACATTGAATTCGGATC |
| SEQ ID NO.5 | BRCA1-2 | TCACAAGCAGCCTTGTTTG |
| SEQ ID NO.6 | BRCA1-P | AGTTGCCTTATTATCGGTATCTTGA |
| SEQ ID NO.7 | RRM1-1 | CAGTGATGTAATGGAAGACCT |
| SEQ ID NO.8 | RRM1-2 | ATTCAGACGATCTTTATTGG |
| SEQ ID NO.9 | RRM1-P | CACATAATGGCAATCACTGTCCC |
| SEQ ID NO.10 | TYMS-1 | CGCTACAGTCTGAGAGATG |
| SEQ ID NO.11 | TYMS-2 | GATCCCTTGATAGACCACAG |
| SEQ ID NO.12 | TYMS-P | ATTCCCTCTGCTGACAACCAAATG |
| SEQ ID NO.13 | GAPDH-1 | GAAGGTGAAGGTCGGAGT |
| SEQ ID NO.14 | GAPDH-2 | GAAGATGGTGATGGGATTTC |
| SEQ ID NO.15 | GAPDH-P | CAAGCTTCCCGTTCTCAGCC |
Wherein ERCC1-P, BRCA1-P, RRM1-P, TYMS-P and GAPDH-P correspond to different fluorescence channels respectively. For example, ERCC1-P corresponds to HEX channel, BRCA1-P corresponds to ROX channel, RRM1-P corresponds to FAM channel, TYMS-P corresponds to CY5 channel, and GAPDH-P corresponds to TAMRA channel.
Example 2
The PCR reaction system was configured according to Table 2.
| Reagent | RT-PCR Buffer | Taq enzyme | Reverse transcriptase | Primer probe |
| Use amount (mu L) | 12.5 | 0.5 | 0.5 | 6.5 |
Wherein the volume of the template was 5. mu. L, so that the volume of the PCR reaction system was 25. mu. L.
The primer probes in table 2 include primers and probes for detecting expression levels of ERCC1, BRCA1, RRM1, TYMS, and primers and probes for detecting the housekeeping gene GAPDH. Wherein, the concentration of the upstream primer and the downstream primer of each target gene (cancer treatment drug related gene) is 0.3 mu M, the concentration of the probe is 0.1 mu M, the concentration of the upstream primer and the downstream primer of the housekeeping gene is 0.15 mu M, and the concentration of the probe is 0.05 mu M. RT-PCR Buffer, Taq enzyme, reverse transcriptase are commercially available reagents, specifically can be purchased from Shanghai worker or general purpose organism or Bao organism.
Can advance mRNA in liver cancer tumor tissues to be used as a template. As a specific method, reference may be made to a method for extracting mRNA from animal tissue in the prior art, which is not described herein again.
The reaction procedure is as follows.
45 ℃ × 15min, 95 ℃ × 2min, (94 ℃ × 10sec +60 ℃ × 40sec) × 40cycles, wherein 45 ℃ × 15min is the reaction stage of reverse transcriptase, the reverse transcription of mRNA into cDNA, 95 ℃ × 2min is the step of reverse transcriptase inactivation, and is also the process of HS Taq enzyme hot start and template DNA pre-denaturation, two-step PCR is adopted, annealing and extension are simultaneously carried out at the stage of 60 ℃ × 40sec, the reaction time is shortened, the temperature adapts to the Tm values of primers and probes, and the amplification efficiency is higher.
The detection results were analyzed as follows.
Because the cycle threshold of the template has a linear relation with the logarithm of the initial copy number of the template, taking the logarithm of the initial copy number of each standard as an abscissa, taking the corresponding cycle threshold as an ordinate, making a corresponding standard curve, analyzing the initial copy number of the target gene contained in the cDNA to be detected according to the cycle threshold of the cDNA to be detected, and reading the initial copy number of the target gene from the marked curve.
According to the scheme provided by the invention, specific primer and probe sequences are designed aiming at the expression of mRNA of different genes (ERCC1, BRCA1, RRM1 and TYMS), and the expression level of mRNA of a plurality of genes (ERCC1, BRCA1, RRM1 and TYMS) can be detected in one tube.
The scheme provided by the invention has the following effects:
1. when the invention is used for detection, the sampling process has higher requirement, and the invention designs an internal standard (housekeeping gene) to monitor sampling, thereby effectively avoiding the problem of false negative possibly caused by sampling;
2. the invention aims at the need of scientific drug-resistant gene typing and individual diagnosis in the treatment process of liver cancer. According to the detection result of the mRNA expression level of the cancer treatment drug related gene in the tumor tissue of the patient, an individualized treatment scheme is formulated, which is beneficial to selecting the chemotherapy drugs suitable for the patient, avoiding ineffective harmful chemotherapy and improving the pertinence of the clinical treatment of the tumor.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A detection reagent for detecting the expression level of a gene related to a cancer treatment drug, wherein the detection reagent is a fluorescent quantitative PCR reagent and comprises at least one of the following components:
a primer pair and a fluorescent probe for detecting ERCC1, a primer pair and a fluorescent probe for detecting BRCA1, a primer pair and a fluorescent probe for detecting RRM1, and a primer pair and a fluorescent probe for detecting TYMS.
2. The detection reagent according to claim 1, wherein the primer pair for detecting ERCC1 consists of the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent probe for detecting ERCC1 is shown in SEQ ID NO. 3.
3. The detection reagent according to claim 1, wherein the primer pair for detecting BRCA1 consists of the sequence shown in SEQ ID No.4 and the sequence shown in SEQ ID No.5, and the nucleotide sequence of the fluorescent probe for detecting BRCA1 is shown in SEQ ID No. 6.
4. The detection reagent according to claim 1, wherein the primer pair for detecting RRM1 consists of a sequence shown in SEQ ID No. 7and a sequence shown in SEQ ID No.8, and the nucleotide sequence of the fluorescent probe for detecting RRM1 is shown in SEQ ID No. 9.
5. The detection reagent according to claim 1, wherein the primer pair for detecting TYMS consists of the sequence shown in SEQ ID NO.10 and the sequence shown in SEQ ID NO.11, and the nucleotide sequence of the fluorescent probe for detecting TYMS is shown in SEQ ID NO. 12.
6. The detection reagent according to claim 1, wherein the cancer is primary liver cancer.
7. A kit for detecting the expression level of a gene related to a liver cancer therapeutic agent, comprising the detection reagent according to any one of claims 1 to 6.
8. A method for detecting the expression level of a gene related to a liver cancer treatment drug comprises the following steps:
extracting mRNA of the tumor tissue;
in the same PCR reaction tube, reverse transcription PCR is carried out by taking mRNA of tumor tissues as a template, and fluorescence quantitative PCR is carried out by taking a reverse transcription product as a template; wherein the primer pair of the reverse transcription PCR and the fluorescence quantitative PCR is at least one of
A primer pair for detecting ERCC1, a primer pair for detecting BRCA1, a primer pair for detecting RRM1 and a primer pair for detecting TYMS.
9. Use of the detection reagent of any one of claims 1-5 in the manufacture of a product for screening personalized therapeutic drugs for cancer patients.
10. The use according to claim 9, wherein the cancer is primary liver cancer.
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