CN103882138A - Multiple-gene detecting kit related to antitumor drugs - Google Patents
Multiple-gene detecting kit related to antitumor drugs Download PDFInfo
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- CN103882138A CN103882138A CN201410135933.1A CN201410135933A CN103882138A CN 103882138 A CN103882138 A CN 103882138A CN 201410135933 A CN201410135933 A CN 201410135933A CN 103882138 A CN103882138 A CN 103882138A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to a multiple-gene detecting kit related to antitumor drugs. The multiple-gene detecting kit comprises a mixture of RT (reverse transcription) primers and a mixture of PCR (polymerase chain reaction) amplification primers, wherein each RT primer and each PCR amplification primer based on genetic groups, reference genes and a reaction internal label are respectively contained in each of the mixture of the RT primers and the mixture of the PCR amplification primers; the multiple-gene detecting kit is characterized in that the genetic groups comprise PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS and TOP2A; the reference genes comprise ACTB, GAPDH and B2M; the reaction internal label is KAN-rRNA (ribosomal RNA). The multiple-gene detecting kit related to antitumor drugs disclosed by the invention can be used for systemically detecting the expression level of a plurality of genes closely related to the antitumor drugs by one step, is simple and convenient to use, good in accuracy and high in detecting efficiency, and can be used for guiding chemotherapy drugs and selecting chemotherapy regimens very well.
Description
Technical field
The present invention relates to a kind of gene detecting kit, the test kit of the multiple antitumor medication related gene expression level of especially a kind of synchronous detection, belongs to Medical Molecular Biology technical field.
Background technology
As everyone knows, chemotherapy is selection scheme the most frequently used in current oncotherapy, but usually curative effect is little after chemotherapy for patient more than half, and same tumour medicine often has very large difference for different patients' curative effect; Toxic and side is larger simultaneously, and it kills and wounds again the cell of healthy tissues in killing tumor cell, sometimes also can develop complications, and many patients doubly groan because of the strong toxic side effect of chemotherapy, even dead.A large amount of clinical research confirmations, rule of thumb treatment tumour, generally only have the curative effect below 30%, and toxic side effect is obvious.The research of pharmacogenetics and pharmacogenomics shows, the difference of different patient's gene expression doses causes the Different therapeutical effect to various antitumor drugs.
The existing article of Nature magazine in 2012 points out, clinical diagnosis transfers to a greater extent according to Molecular Detection result by depending on traditionally pathological diagnosis mode, and Molecular Detection has become the necessary detection means of clinical diagnosis.High-caliber Molecular Detection means and product are for the individualized treatment of New Times is paved the way.Depend on pharmacogenetics and the pharmacogenomics newest research results at aspects such as antitumor drug mechanism of action, utilize the significant correlation mechanism of action effect and the related gene expression of some antitumor drug, if our energy before the selection of carrying out chemotherapy regimen is convenient, fast and exactly patient's genes involved is carried out to system detection, can significantly improve curative effect of medication and alleviate toxic side effect, and effectively strive for treatment time, finally make patient in curative effect and benefit economically.
Comprise with the most closely-related gene of antitumor medication at present, PTEN(phosphatase and tensin homolog, homology Phosphoric acid esterase-tensin), EGFR(epidermal growth factor receptor, EGF-R ELISA), DPYD(dihydropyrimide dehydrogenase, dihydropyrimidine dehydrogenase), HER2(human epidermalgrowth factor receptor-2, ErbB-2), RRM1(ribonucleotide reductase M1, ribose nucleotide reducing ferment M 1), ERCC1 (excision repair cross complementation 1, Excision repair cross-completion 1 gene), TUBB3(tubulin-beta 3, beta-tubulin 3), TOP1(topoisomerase I, topoisomerase 1), TYMS(thymidylate synthetase, thymidylate synthetase), TOP2A(topoisomerase DNA II alpha, topoisomerase 2) etc., the height of these target genes mRNA level can instruct such as Trastuzumab, Gefitinib, fluorine class, the selection of the Treated with Chemotherapeutic Drugs things such as anti-microtubule class, to implement individualized treatment scheme, improves curative effect.
The common method of gene expression detection situation has at present: FISH, immunohistochemical methods, quantitative fluorescent PCR (qPCR), gene chip etc.Wherein, FISH uses fluorescently-labeled probe to go to detect the rna expression situation on tissue slice; Immunohistochemical methods is to use antibody to go to detect the protein expression situation on tissue slice.But the shortcoming of these two kinds of methods is all cannot be quantitative, and experimental result must be observed and subjective judgement by experienced detection person.In addition, biochip technology can detect thousands of the even transcriptional levels of up to ten thousand genes conventionally simultaneously, but be often applied to the analysis of full gene expression profile, the expression level analysis meeting of carrying out some specific gene group by high-throughout method like this causes cost too high, be difficult to practical requirement.And quantitative fluorescent PCR (qPCR) is the routine techniques that detects rna expression, it is also the most frequently used technology that detects tumor tissues expression conditions.If but need to detect the expression of multiple genes, mostly need to detect each gene one by one, and be difficult to arrange reference gene, flux is low, and cost is high, convenience is poor.
In a reaction system, apply multipair primer and carry out pcr amplification to detecting multiple genes, carry out multiple gene test, need repeatedly amplification condition be optimized and be adjusted and checking, and need select and verify suitable primer sequence, if there is the testing product of ripe application to save a large amount of man power and materials.
Summary of the invention
The object of this invention is to provide one easy to use, accuracy is good, and the multiple gene detecting kit relevant to antitumor medication that detection efficiency is high can the disposable expression level that systematically detects the closely related gene of multiple and antitumor medication.
The present invention is that a kind of technical scheme that solves the problems of the technologies described above proposition is: the multiple gene detecting kit that a kind of and antitumor medication is relevant, comprise the mixture of RT primer and the mixture of pcr amplification primer, the mixture of described RT primer (reverse transcription primer) comprises based on gene group, the each RT primer of target in reference gene and reaction, the mixture of described pcr amplification primer comprises based on corresponding gene group, the each pcr amplification primer of target in reference gene and reaction, described gene group comprises PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS and TOP2A, described reference gene comprises ACTB, GAPDH and B2M, in described reaction, mark is KAN-r RNA.
Above-mentioned each pcr amplification primer sequence is made up of the forward universal primer sequence of specific primer sequence and 5 ' end, and described each RT primer sequence is made up of the reverse universal primer sequence of specific primer sequence and 5 ' end;
The specific primer sequence of the pcr amplification primer based on described PTEN is as shown in SEQ ID No.1, and the specific primer sequence of RT primer is as shown in SEQ ID No.2;
The specific primer sequence of the pcr amplification primer based on described EGFR is as shown in SEQ ID No.3, and the specific primer sequence of RT primer is as shown in SEQ ID No.4;
The specific primer sequence of the pcr amplification primer based on described DPYD is as shown in SEQ ID No.5, and the specific primer sequence of RT primer is as shown in SEQ ID No.6;
The specific primer sequence of the pcr amplification primer based on described HER2 is as shown in SEQ ID No.7, and the specific primer sequence of RT primer is as shown in SEQ ID No.8;
The specific primer sequence of the pcr amplification primer based on described RRM1 is as shown in SEQ ID No.9, and the specific primer sequence of RT primer is as shown in SEQ ID No.10;
The specific primer sequence of the pcr amplification primer based on described ERCC1 is as shown in SEQ ID No.11, and the specific primer sequence of RT primer is as shown in SEQ ID No.12;
The specific primer sequence of the pcr amplification primer based on described TUBB3 is as shown in SEQ ID No.13, and the specific primer sequence of RT primer is as shown in SEQ ID No.14;
The specific primer sequence of the pcr amplification primer based on described TOP1 is as shown in SEQ ID No.15, and the specific primer sequence of RT primer is as shown in SEQ ID No.16;
The specific primer sequence of the pcr amplification primer based on described TYMS is as shown in SEQ ID No.17, and the specific primer sequence of RT primer is as shown in SEQ ID No.18;
The specific primer sequence of the pcr amplification primer based on described TOP2A is as shown in SEQ ID No.19, and the specific primer sequence of RT primer is as shown in SEQ ID No.20;
The specific primer sequence of the pcr amplification primer based on described ACTB is as shown in SEQ ID No.21, and the specific primer sequence of RT primer is as shown in SEQ ID No.22;
The specific primer sequence of the pcr amplification primer based on described GAPDH is as shown in SEQ ID No.23, and the specific primer sequence of RT primer is as shown in SEQ ID No.24;
The specific primer sequence of the pcr amplification primer based on described B2M is as shown in SEQ ID No.25, and the specific primer sequence of RT primer is as shown in SEQ ID No.26;
The specific primer sequence of the pcr amplification primer based on described KAN-r is as shown in SEQ ID No.27, and the specific primer sequence of RT primer is as shown in SEQ ID No.28.
Technique scheme is preferably: the concentration of the pcr amplification primer based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene in reaction system is 150nM～250nM, and the concentration of the RT primer based on PTEN gene in reaction system is 10nM～20nM; The concentration of RT primer based on EGFR gene in reaction system is 1800nM～2200nM; The concentration of RT primer based on DPYD, RRM1, TYMS and KAN-r gene in reaction system is 450nM～550nM; The concentration of RT primer based on HER2 gene in reaction system is 50nM～70nM; The concentration of RT primer based on ERCC1 and TOP1 gene in reaction system is 200nM～300nM; The concentration of RT primer based on TUBB3 and TOP2A gene in reaction system is 800nM～1200nM; The concentration of RT primer based on ACTB gene in reaction system is 20nM～40nM; The concentration of RT primer based on GAPDH and B2M gene in reaction system is 8nM～12nM.
Technique scheme further preferably: the concentration of the pcr amplification primer based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene in reaction system is 200nM, and the concentration of the RT primer based on PTEN gene in reaction system is 15nM; The concentration of RT primer based on EGFR gene in reaction system is 2000nM; The concentration of RT primer based on DPYD, RRM1, TYMS and KAN-r gene in reaction system is 500nM; The concentration of RT primer based on HER2 gene in reaction system is 60nM; The concentration of RT primer based on ERCC1 and TOP1 gene in reaction system is 250nM; The concentration of RT primer based on TUBB3 and TOP2A gene in reaction system is 1000nM; The concentration of RT primer based on ACTB gene in reaction system is 30nM; The concentration of RT primer based on GAPDH and B2M gene in reaction system is 10nM.
The multiple gene detecting kit that above-mentioned and antitumor medication is relevant, also comprises forward universal primer and the reverse mixture of universal primer; Described forward universal primer is fluorescent dye primer; Described forward universal primer sequence is as shown in SEQ ID No.29, and described reverse universal primer sequence is as shown in SEQ ID No.30; Described forward universal primer and the oppositely concentration of universal primer in reaction system are 1.0 μ M～1.5 μ M.
The fluorescent mark of above-mentioned forward universal primer comprises Cy5, Cy3 or FAM.
Technique scheme is preferably: above-mentioned forward universal primer and the oppositely concentration of universal primer in reaction system are 1.2 μ M.
The multiple gene detecting kit that above-mentioned and antitumor medication is relevant also comprises ThermoScript II, reverse transcription damping fluid, KAN-r RNA, archaeal dna polymerase, PCR damping fluid, dNTP and ultrapure water.
The multiple gene detecting kit that above-mentioned and antitumor medication is relevant also comprises positive reference substance, and described positive reference substance is the RNA mixture extracting from tumour cell, and described positive reference substance comprises target mRNA in described gene group, reference gene and reaction.It can also be the mixture of the DNA fragmentation based on target pcr amplification primer in described gene group, reference gene and reaction and RT primer clone gained as the positive reference substance of the reacted PCR reaction of RT.
The application of the multiple gene detecting kit that above-mentioned and antitumor medication is relevant is to instruct chemotherapy medication, selects chemotherapy regimen.
The present invention has positive effect:
(1) multiple gene detecting kit of the present invention adopts KAN-r can effectively monitor the normal work of reaction system as mark in reaction, get rid of the error that Initial R NA template amount difference is brought, and mark as in reacting with carrier etc. in traditional method, thereby easily cause its efficient deviation that increases and affect effective amplification of target gene and cause result to judge; The present invention also adopts three reference genes as PCR reaction contrast, the expression level of 10 goal gene that detect will carry out homogenization by these two reference genes, overcome the deviation causing due to unequal amplification in normal PCR, realized the expression of one group of goal gene of accurate quantification.Above-mentioned internal reference and interior target arrange capillary electrophoresis and the detection technique of fluorescence in conjunction with GeXP, be different from conventional gel electrophoretic analysis pattern, make PCR interpretation of result more directly perceived, succinct, reliable, be easy to identification judgement, more be conducive to normalizing operation, realized hypersensitivity and at utmost reduced false positive problem.
(2) multiple gene detecting kit of the present invention is optimized reaction system, mainly each primer sequence has been carried out to repeatedly screening and has avoided as far as possible the interference problem between primer; Repeatedly adjust the suitable concn of each primer, optimized reaction system.Multiple gene detecting kit of the present invention utilizes the combination of fluorescent mark universal primer for having realized a PCR system inter-sync systematize and detected ten goal gene sites with the special primer of closely-related ten genes of antitumor medication, realized detect quick and convenient, accuracy has also obtained checking.
(3) multiple gene detecting kit of the present invention has utilized GenomeLab GeXP genetic analysis systems high-throughput and automatization advantage; adopt 96 hole loading systems to carry out multiple sample mass-producing detection; simple operation, working conditions can medelling, is convenient to large-scale promotion application.The testing cost of each sample is in 50 yuan.Realized high-throughput, low cost, for system medication guide has been striven for the time.
(4) multiple gene test of the present invention; to carrying out the product development of synchronous detection with closely-related 10 genes of antitumor medication; the application of the column criterion of going forward side by side and mass-producing; there is the features such as flux is high, cost is low, convenient, reproducible, accuracy is good, for the successful development of the dissimilar test kit of adjusting required detection target gene provides reference.
Accompanying drawing explanation
Fig. 1 is that the RNA mixture that adopts test kit of the present invention to extract kinds of tumor cells carries out RT, PCR and utilizes GeXP system to carry out the collection of illustrative plates after capillary electrophoresis analysis.
Embodiment
Below by embodiment, the present invention is specifically described; be necessary to be pointed out that at this following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, person skilled in art can make some nonessential improvement and adjustment to the present invention according to the invention described above content.The experimental technique of unreceipted actual conditions in literary composition, the condition described in " molecular cloning experiment guide " book that the Science Press of conventionally writing as J. Pehanorm Brooker etc. according to normal condition publishes for 2002, or the condition of advising according to manufacturers.Unless otherwise defined, the same meaning that all specialties that use in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.
One, the composition of test kit.
The multiple gene detecting kit that the present invention is relevant to antitumor medication comprises the following test tube being fixed in paper package box:
1) RT enzyme (ThermoScript II);
2) RT damping fluid (reverse transcription damping fluid);
3) mixture of RT primer (mixture of reverse transcription primer);
4）KAN-r RNA；
5) archaeal dna polymerase;
6) PCR damping fluid (magnesium chloride containing);
7）dNTP；
8) mixture of pcr amplification primer;
9) forward universal primer and the oppositely mixture of universal primer;
10) positive reference substance;
11) ultrapure water (without RNA enzyme/DNA enzyme).
Each pcr amplification primer 5 ' end is provided with forward universal primer sequence, and 5 ' end of each RT primer is provided with reverse universal primer sequence.
The sequence of forward universal primer is: AGGTGACACTATAGAATA.
Oppositely the sequence of universal primer is: GTACGACTCACTATAGGGA.
On forward universal primer with Cy5 fluorescent mark.
Positive reference substance is the RNA mixture extracting from kinds of tumor cells, comprises target mRNA in described gene group, reference gene and reaction.
The sequence of RT primer and pcr amplification primer according to gene group in the mRNA sequence of each gene design across exon.Every pair of amplimer can amplify the PCR fragment of length-specific.The amplification theoretical fragment length that different genes is corresponding is as shown in table 1, and actual fragment length may be slightly different.
In the mixture of the mixture of RT primer and pcr amplification primer, the concentration of each RT primer and pcr amplification primer is as shown in table 1.Forward universal primer and oppositely universal primer concentration are far above the concentration of RT primer and pcr amplification primer.Preferably the concentration of forward universal primer and reverse universal primer is 1.2 μ M.
Table 1 primer mark sheet
Two, the purposes of test kit.
The multiple gene detecting kit that of the present invention and antitumor medication is relevant is used for instructing chemotherapy medication according to the expression level of ten kinds of antitumor genes involveds, selects chemotherapy regimen, and the purposes of the expression level of each genes involved is as follows:
(1) the curative effect of medication positive correlation of PTEN expression level and Erbitux/Trastuzumab.
(2) EGFR expression level with the positive correlation of Gefitinib/Tarceva/Cetuximab/Victibix curative effect.
(3) the curative effect of medication negative correlation of DPYD expression level and 5-Fu.
(4) the curative effect of medication positive correlation of HER2 expression level and Trastuzumab.
(5) RRM1 expression level and gemcitabine curative effect of medication negative correlation.
(6) ERCC1 expression level and therapeutic effectiveness of platinum medicaments negative correlation.
(7) the curative effect of medication negative correlation of TUBB3 expression level and anti-microtubule class medicine.
(8) TOP1 expression level and the positive correlation of irinotecan curative effect of medication.
(9) TYMS with fluorine class/pemetrexed/capecitabine curative effect negative correlation.
(10) TOP2A expression level and the positive correlation of anthracene nucleus medicament curative effect.
Three, the using method of test kit.
The multiple gene detecting kit that of the present invention and antitumor medication is relevant detects in the paraffin organization section sample of tumour patient the expression level with closely-related ten goal gene of antitumor medication, and concrete detecting step is as follows:
1, collecting sample extracts RNA from the paraffin organization section of tumour patient.
Paraffin-embedded tissue is cut into 10 μ m thin slices, gets 5 of thin slices, use paraffin RNA to extract test kit, extract RNA.After ultraviolet spectrophotometer is measured concentration, 4 ℃ or-20 ℃ save backup.
2, take the tumor tissues RNA that extracts as template, reverse transcription becomes cDNA.
The mixture that uses RT primer, becomes cDNA by step 1 gained RNA reverse transcription.The component of the RT reaction solution of preparing is as shown in table 2.
Table 2 RT reaction solution component list
The reaction conditions of reverse transcription is: 48 ℃ of 1min, and 42 ℃ of 60min, 95 ℃ of 5min, remain on 4 ℃ until collect RT product.Total time, about 75min. reverse transcription product can be preserved the several months at-20 ℃.
3,, take cDNA as template, carry out multi-PRC reaction.
The cDNA that reverse transcription in step 2 is obtained is template, uses pcr amplification primer, forward universal primer and reverse universal primer, amplification PCR product.The composition of the PCR reaction solution of preparing is as shown in table 3.
Table 3 PCR reaction solution component list
Reaction system after preparing is moved following program on PCR reaction instrument:
（1）95℃ 10min；
（2）94℃ 30sec；
（3）55℃ 30sec；
（4）72℃ 30sec；
(5) repeat the circulation of 34 of (2)～(4);
（6）72℃ 5min；
(7) 4 ℃ of insulations.
4, use GenomeLab GeXP genetic analyzer, carry out capillary electrophoresis, detect fluorescent signal, go forward side by side line number according to one's analysis.
In every hole of 96 hole sample panel, add sample-loading buffer (methane amide, SLS) 38.5 μ L, interior mark Marker(DNA Standard 400) 0.5 μ L, multiple PCR products 1 μ L, adds 1 mineral oil after fully mixing, prevent methane amide oxidation.
In every hole of another 96 hole damping fluid plate, add the GenomeLab dissociating buffer of approximately 250 μ L.
According to the operational manual of GeXP genetic analyzer, kapillary and gel are installed.Sample panel and damping fluid plate are put into machine, operation Frag-3 separable programming.Carry out the GeXP analytical procedure of acquiescence, last save data.As shown in Figure 1, wherein X-coordinate represents fragment length to the capillary electrophoresis peak figure that experiment obtains, and ordinate zou represents fluorescence intensity.
Use GeXP acquiescence analytical standard " Default GeXP Analysis Parameters ", carry out fragment analysis.
Fragment analysis result is imported to the software kit eXpress Profiler of GeXP system.Select Kan-r RNA as mark in reaction, select ACTB and GAPDH as reference gene, obtain the expression level of the each gene after homogenization.Homogenization can be eliminated by different the brought errors different from detection reaction efficiency of Initial R NA template amount.The peak collection of illustrative plates of gained, X-coordinate represents amplified fragments size, ordinate zou expression signal power.According to each gene amplification the calculated by peak area at corresponding peak obtain relative expression's level of antitumor medication genes involved.Fig. 1 is that the RNA mixture that adopts test kit of the present invention to extract kinds of tumor cells carries out RT, PCR and utilizes GeXP system to carry out the collection of illustrative plates after capillary electrophoresis analysis, and substance gene test result is basically identical with carrying out.That gene expression dose with normal people is as reference to judging of result.
Four, the sensitivity of test kit and specificity analyses.
Sensitivity analysis: by after positive reference substance doubling dilution, carry out pcr amplification, then use GenomeLab GeXP genetic analyzer to carry out capillary electrophoresis, until almost can't detect fluorescent signal, this value is lowest detection line, and the sensitivity that is test kit is 1.0ng.
Specificity analyses: positive reference substance uses GenomeLab GeXP genetic analyzer to carry out capillary electrophoresis analysis after substance pcr amplification, detects unimodal into target fragment size.
Obviously, above-described embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And these belong to apparent variation that spirit of the present invention extended out or variation still among protection scope of the present invention.
Claims (10)
1. a multiple gene detecting kit relevant to antitumor medication, comprise the mixture of RT primer and the mixture of pcr amplification primer, the mixture of described RT primer comprises based on the each RT primer of target in gene group, reference gene and reaction, the mixture of described pcr amplification primer comprises based on the each pcr amplification primer of target in corresponding gene group, reference gene and reaction, it is characterized in that: described gene group comprises PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS and TOP2A; Described reference gene comprises ACTB, GAPDH and B2M; In described reaction, mark is KAN-r RNA.
2. a kind of multiple gene detecting kit relevant with antitumor medication according to claim 1, it is characterized in that: described each pcr amplification primer sequence is made up of the forward universal primer sequence of specific primer sequence and 5 ' end, described each RT primer sequence is made up of the reverse universal primer sequence of specific primer sequence and 5 ' end;
The specific primer sequence of the pcr amplification primer based on described PTEN is as shown in SEQ ID No.1, and the specific primer sequence of RT primer is as shown in SEQ ID No.2;
The specific primer sequence of the pcr amplification primer based on described EGFR is as shown in SEQ ID No.3, and the specific primer sequence of RT primer is as shown in SEQ ID No.4;
The specific primer sequence of the pcr amplification primer based on described DPYD is as shown in SEQ ID No.5, and the specific primer sequence of RT primer is as shown in SEQ ID No.6;
The specific primer sequence of the pcr amplification primer based on described HER2 is as shown in SEQ ID No.7, and the specific primer sequence of RT primer is as shown in SEQ ID No.8;
The specific primer sequence of the pcr amplification primer based on described RRM1 is as shown in SEQ ID No.9, and the specific primer sequence of RT primer is as shown in SEQ ID No.10;
The specific primer sequence of the pcr amplification primer based on described ERCC1 is as shown in SEQ ID No.11, and the specific primer sequence of RT primer is as shown in SEQ ID No.12;
The specific primer sequence of the pcr amplification primer based on described TUBB3 is as shown in SEQ ID No.13, and the specific primer sequence of RT primer is as shown in SEQ ID No.14;
The specific primer sequence of the pcr amplification primer based on described TOP1 is as shown in SEQ ID No.15, and the specific primer sequence of RT primer is as shown in SEQ ID No.16;
The specific primer sequence of the pcr amplification primer based on described TYMS is as shown in SEQ ID No.17, and the specific primer sequence of RT primer is as shown in SEQ ID No.18;
The specific primer sequence of the pcr amplification primer based on described TOP2A is as shown in SEQ ID No.19, and the specific primer sequence of RT primer is as shown in SEQ ID No.20;
The specific primer sequence of the pcr amplification primer based on described ACTB is as shown in SEQ ID No.21, and the specific primer sequence of RT primer is as shown in SEQ ID No.22;
The specific primer sequence of the pcr amplification primer based on described GAPDH is as shown in SEQ ID No.23, and the specific primer sequence of RT primer is as shown in SEQ ID No.24;
The specific primer sequence of the pcr amplification primer based on described B2M is as shown in SEQ ID No.25, and the specific primer sequence of RT primer is as shown in SEQ ID No.26;
The specific primer sequence of the pcr amplification primer based on described KAN-r is as shown in SEQ ID No.27, and the specific primer sequence of RT primer is as shown in SEQ ID No.28.
3. a kind of multiple gene detecting kit relevant with antitumor medication according to claim 2, it is characterized in that: the concentration of the pcr amplification primer based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene in reaction system is 150nM～250nM, the concentration of the RT primer based on PTEN gene in reaction system is 10nM～20nM; The concentration of RT primer based on EGFR gene in reaction system is 1800nM～2200nM; The concentration of RT primer based on DPYD, RRM1, TYMS and KAN-r gene in reaction system is 450nM～550nM; The concentration of RT primer based on HER2 gene in reaction system is 50nM～70nM; The concentration of RT primer based on ERCC1 and TOP1 gene in reaction system is 200nM～300nM; The concentration of RT primer based on TUBB3 and TOP2A gene in reaction system is 800nM～1200nM; The concentration of RT primer based on ACTB gene in reaction system is 20nM～40nM; The concentration of RT primer based on GAPDH and B2M gene in reaction system is 8nM～12nM.
4. a kind of multiple gene detecting kit relevant with antitumor medication according to claim 3, it is characterized in that: the concentration of the pcr amplification primer based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene in reaction system is 200nM, the concentration of the RT primer based on PTEN gene in reaction system is 15nM; The concentration of RT primer based on EGFR gene in reaction system is 2000nM; The concentration of RT primer based on DPYD, RRM1, TYMS and KAN-r gene in reaction system is 500nM; The concentration of RT primer based on HER2 gene in reaction system is 60nM; The concentration of RT primer based on ERCC1 and TOP1 gene in reaction system is 250nM; The concentration of RT primer based on TUBB3 and TOP2A gene in reaction system is 1000nM; The concentration of RT primer based on ACTB gene in reaction system is 30nM; The concentration of RT primer based on GAPDH and B2M gene in reaction system is 10nM.
5. according to a kind of relevant with the antitumor medication multiple gene detecting kit one of claim 1 to 4 Suo Shu, it is characterized in that: the mixture that also comprises forward universal primer and reverse universal primer; Described forward universal primer is fluorescent dye primer; Described forward universal primer sequence is as shown in SEQ ID No.29, and described reverse universal primer sequence is as shown in SEQ ID No.30; Described forward universal primer and the oppositely concentration of universal primer in reaction system are 1.0 μ M～1.5 μ M.
6. a kind of multiple gene detecting kit relevant with antitumor medication according to claim 5, is characterized in that: the fluorescent mark of described forward universal primer comprises Cy5, Cy3 or FAM.
7. a kind of multiple gene detecting kit relevant with antitumor medication according to claim 5, is characterized in that: described forward universal primer and the oppositely concentration of universal primer in reaction system are 1.2 μ M.
8. according to a kind of relevant with the antitumor medication multiple gene detecting kit one of claim 1 to 4 Suo Shu, it is characterized in that: also comprise ThermoScript II, reverse transcription damping fluid, KAN-r RNA, archaeal dna polymerase, PCR damping fluid, dNTP and ultrapure water.
9. according to a kind of relevant with the antitumor medication multiple gene detecting kit one of claim 1 to 4 Suo Shu, it is characterized in that: also comprise positive reference substance, described positive reference substance is the RNA mixture extracting from several tumour cells, and described positive reference substance comprises target mRNA in described gene group, reference gene and reaction.
10. an application for the multiple gene detecting kit relevant to antitumor medication described in the claims 1, is characterized in that: instruct chemotherapy medication, select chemotherapy regimen.
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