CN103882138B - A kind of multiple gene detection kit relevant to anti-tumor drug - Google Patents
A kind of multiple gene detection kit relevant to anti-tumor drug Download PDFInfo
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Abstract
The present invention relates to a kind of multiple gene detection kit relevant to anti-tumor drug, comprise the mixture of RT primer and the mixture of pcr amplification primer, comprise respectively based on target each RT primer in gene group, reference gene and reaction and each pcr amplification primer in the mixture of RT primer and the mixture of pcr amplification primer, it is characterized in that: gene group comprises PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS and TOP2A; Reference gene comprises ACTB, GAPDH and B2M; In reaction, mark is KAN-r RNA.The multiple gene detection kit relevant to anti-tumor drug of the present invention disposablely systematically can detect expression level that is multiple and the closely related gene of anti-tumor drug, easy to use, accuracy is good, and detection efficiency is high, can perform well in instructing chemotherapy medication, select chemotherapy regimen.
Description
Technical field
The present invention relates to a kind of gene detecting kit, the test kit of especially a kind of synchronous detection multiple anti-tumor drug related gene expression level, 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 the curative effect of different patient; Toxic and side is comparatively large simultaneously, and it is while killing tumor cell, kills and wounds again the cell of healthy tissues, 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 confirmation, rule of thumb treatment tumour, generally only have the curative effect of less than 30%, and toxic side effect is obvious.The research of pharmacogenetics and pharmacogenomics shows, the difference of different patient's gene expression dose causes the Different therapeutical effect to various antitumor drug.
Nature magazine in 2012 has article and points out, clinical diagnosis transfers to a greater extent according to Molecular Detection result by depending on pathological diagnosis mode traditionally, 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 newest research results of pharmacogenomics in antitumor drug mechanism of action etc., utilize the action effect of some antitumor drug and the significant correlation mechanism of related gene expression, if we can be convenient, fast and carry out systems axiol-ogy to the genes involved of patient exactly before the selection carrying out chemotherapy regimen, can curative effect of medication be significantly improved and alleviate toxic side effect, and effectively strive for treatment time, finally make patient in curative effect and benefit economically.
Current and the most closely-related gene of anti-tumor drug comprises, 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 gene mRNA level in-site 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 current gene expression detection situation has: 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 uses 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 carry out observation and subjective judgement by experienced detection person.In addition, biochip technology can detect thousands of the even transcriptional level of up to ten thousand genes usually simultaneously, but be often applied to the analysis of full genome express spectra, the expression level analysis meeting 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 detecting rna expression, it is also the most frequently used technology detecting tumor tissues expression conditions.If but need the expression detecting multiple gene, then 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, applying multipair primer carrying out pcr amplification to detecting multiple gene, namely multiple gene detection is carried out, repeatedly need be optimized adjustment and checking to amplification condition, 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 a kind of easy to use, accuracy is good, the multiple gene detection kit relevant to anti-tumor drug that detection efficiency is high, disposablely can systematically detect expression level that is multiple and the closely related gene of anti-tumor drug.
The present invention is a kind of technical scheme solving the problems of the technologies described above proposition: a kind of multiple gene detection kit relevant to anti-tumor drug, 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, reference gene and the interior each RT primer of target of reaction, the mixture of described pcr amplification primer comprises based on corresponding gene group, reference gene and the interior each pcr amplification primer of target of 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.
The forward universal primer sequence that above-mentioned each pcr amplification primer sequence is held by specific primer sequence and 5 ' forms, and the reverse universal primer sequence that described each RT primer sequence is held by specific primer sequence and 5 ' forms;
Based on the specific primer sequence of the pcr amplification primer of described PTEN as shown in SEQ ID No.1, the specific primer sequence of RT primer is as shown in SEQ ID No.2;
Based on the specific primer sequence of the pcr amplification primer of described EGFR as shown in SEQ ID No.3, the specific primer sequence of RT primer is as shown in SEQ ID No.4;
Based on the specific primer sequence of the pcr amplification primer of described DPYD as shown in SEQ ID No.5, the specific primer sequence of RT primer is as shown in SEQ ID No.6;
Based on the specific primer sequence of the pcr amplification primer of described HER2 as shown in SEQ ID No.7, the specific primer sequence of RT primer is as shown in SEQ ID No.8;
Based on the specific primer sequence of the pcr amplification primer of described RRM1 as shown in SEQ ID No.9, the specific primer sequence of RT primer is as shown in SEQ ID No.10;
Based on the specific primer sequence of the pcr amplification primer of described ERCC1 as shown in SEQ ID No.11, the specific primer sequence of RT primer is as shown in SEQ ID No.12;
Based on the specific primer sequence of the pcr amplification primer of described TUBB3 as shown in SEQ ID No.13, the specific primer sequence of RT primer is as shown in SEQ ID No.14;
Based on the specific primer sequence of the pcr amplification primer of described TOP1 as shown in SEQ ID No.15, the specific primer sequence of RT primer is as shown in SEQ ID No.16;
Based on the specific primer sequence of the pcr amplification primer of described TYMS as shown in SEQ ID No.17, the specific primer sequence of RT primer is as shown in SEQ ID No.18;
Based on the specific primer sequence of the pcr amplification primer of described TOP2A as shown in SEQ ID No.19, the specific primer sequence of RT primer is as shown in SEQ ID No.20;
Based on the specific primer sequence of the pcr amplification primer of described ACTB as shown in SEQ ID No.21, the specific primer sequence of RT primer is as shown in SEQ ID No.22;
Based on the specific primer sequence of the pcr amplification primer of described GAPDH as shown in SEQ ID No.23, the specific primer sequence of RT primer is as shown in SEQ ID No.24;
Based on the specific primer sequence of the pcr amplification primer of described B2M as shown in SEQ ID No.25, the specific primer sequence of RT primer is as shown in SEQ ID No.26;
Based on the specific primer sequence of the pcr amplification primer of described KAN-r as shown in SEQ ID No.27, the specific primer sequence of RT primer is as shown in SEQ ID No.28.
Being preferably of technique scheme: the concentration of pcr amplification primer in reaction system based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene is 150nM ~ 250nM, and the concentration of RT primer in reaction system based on PTEN gene is 10nM ~ 20nM; The concentration of RT primer in reaction system based on EGFR gene is 1800nM ~ 2200nM; The concentration of RT primer in reaction system based on DPYD, RRM1, TYMS and KAN-r gene is 450nM ~ 550nM; The concentration of RT primer in reaction system based on HER2 gene is 50nM ~ 70nM; The concentration of RT primer in reaction system based on ERCC1 and TOP1 gene is 200nM ~ 300nM; The concentration of RT primer in reaction system based on TUBB3 and TOP2A gene is 800nM ~ 1200nM; The concentration of RT primer in reaction system based on ACTB gene is 20nM ~ 40nM; The concentration of RT primer in reaction system based on GAPDH and B2M gene is 8nM ~ 12nM.
Being preferably further of technique scheme: the concentration of pcr amplification primer in reaction system based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene is 200nM, and the concentration of RT primer in reaction system based on PTEN gene is 15nM; The concentration of RT primer in reaction system based on EGFR gene is 2000nM; The concentration of RT primer in reaction system based on DPYD, RRM1, TYMS and KAN-r gene is 500nM; The concentration of RT primer in reaction system based on HER2 gene is 60nM; The concentration of RT primer in reaction system based on ERCC1 and TOP1 gene is 250nM; The concentration of RT primer in reaction system based on TUBB3 and TOP2A gene is 1000nM; The concentration of RT primer in reaction system based on ACTB gene is 30nM; The concentration of RT primer in reaction system based on GAPDH and B2M gene is 10nM.
The above-mentioned multiple gene detection kit relevant to anti-tumor drug, also comprises the mixture of 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 concentration of reverse universal primer in reaction system are 1.0 μMs ~ 1.5 μMs.
The fluorescent mark of above-mentioned forward universal primer comprises Cy5, Cy3 or FAM.
Being preferably of technique scheme: above-mentioned forward universal primer and the concentration of reverse universal primer in reaction system are 1.2 μMs.
The above-mentioned multiple gene detection kit relevant to anti-tumor drug also comprises ThermoScript II, reverse transcription buffer, KAN-r RNA, archaeal dna polymerase, PCR damping fluid, dNTP and ultrapure water.
The above-mentioned multiple gene detection kit relevant to anti-tumor drug also comprises positive reference substance, and described positive reference substance is the RNA mixture extracted from tumour cell, and described positive reference substance comprises target mRNA in described gene group, reference gene and reaction.The positive reference substance reacted as the reacted PCR of RT can also be the mixture of cloning the DNA fragmentation of gained based on target pcr amplification primer in described gene group, reference gene and reaction and RT primer.
Chemotherapy medication is instructed in the application of the above-mentioned multiple gene detection kit relevant to anti-tumor drug, selects chemotherapy regimen.
The present invention has positive effect:
(1) multiple gene detection kit of the present invention adopts KAN-r effectively can 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 in traditional method with carrier etc. as mark in reaction, easily cause its efficient amplification thus affect effective amplification of target gene and the deviation that causes result to judge; The present invention also adopts three reference genes as PCR reaction contrast, the expression level of 10 goal gene detected will carry out homogenization by these two reference genes, to overcome in normal PCR due to the deviation that unequal amplification causes, realize the expression of accurate quantification one group of goal gene.Above-mentioned internal reference and interior target arrange capillary electrophoresis in conjunction with GeXP and detection technique of fluorescence, be different from conventional gel electrophoretic analysis pattern, make PCR interpretation of result more directly perceived, succinct, reliable, be easy to identify and judge, more be conducive to normalizing operation, achieve hypersensitivity and at utmost reduce false positive issue.
(2) multiple gene detection kit of the present invention is optimized reaction system, has mainly carried out repeatedly screening the interference problem avoided as far as possible between primer to each primer sequence; Repeatedly adjust the suitable concn of each primer, optimize reaction system.Multiple gene detection kit of the present invention utilizes the combination of fluorescent mark universal primer to achieve for the special primer with anti-tumor drug closely-related ten genes and detects ten goal gene sites a PCR system inter-sync systematize, achieve the quick and convenient of detection, accuracy have also been obtained checking.
(3) multiple gene detection kit of the present invention make use of GenomeLab GeXP genetic analysis systems high-throughput and automatization advantage; 96 hole loading systems are adopted 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.Achieve high-throughput, low cost, strive for the time for Systemic administration instructs.
(4) multiple gene of the present invention detects; the product development of synchronous detection is carried out to closely-related 10 genes of anti-tumor drug; the application of 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, providing reference for adjusting the required successful development detecting the dissimilar test kit of target gene.
Accompanying drawing explanation
Fig. 1 adopts test kit of the present invention carry out RT, PCR to the RNA mixture that kinds of tumor cells is extracted and utilize GeXP system to carry out the collection of illustrative plates after capillary electrophoresis analysis.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that 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 according to the invention described above content to the present invention.The experimental technique of unreceipted actual conditions in literary composition, the condition described in " Molecular Cloning: A Laboratory guide " book that the Science Press that conveniently condition is write as J. Pehanorm Brooker etc. usually publishes for 2002, or according to the condition that manufacturers advises.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.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 detection kit that the present invention is relevant to anti-tumor drug comprises the following test tube be fixed in paper package box:
1) RT enzyme (ThermoScript II);
2) RT damping fluid (reverse transcription buffer);
3) mixture (mixture of reverse transcription primer) of RT 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) mixture of forward universal primer and reverse universal primer;
10) positive reference substance;
11) ultrapure water (without RNA enzyme/DNA enzymatic).
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.
The sequence of reverse universal primer is: GTACGACTCACTATAGGGA.
With Cy5 fluorescent mark on forward universal primer.
Positive reference substance is the RNA mixture extracted 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 designs across exon according to the mRNA sequence of each gene in gene group.Often 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 RT primer and the mixture of pcr amplification primer, the concentration of each RT primer and pcr amplification primer is as shown in table 1.Forward universal primer and reverse universal primer concentration are far above the concentration of RT primer and pcr amplification primer.The concentration of preferred forward universal primer and reverse universal primer is 1.2 μMs.
Table 1 primer mark sheet
Two, the purposes of test kit.
The multiple gene detection kit relevant to anti-tumor drug of the present invention is for instructing chemotherapy medication according to the expression level of ten kinds of antitumor genes involveds, and select chemotherapy regimen, 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 Gefitinib/Tarceva/Cetuximab/Victibix curative effect positive correlation.
(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 detection kit relevant to anti-tumor drug of the present invention detects the expression level with anti-tumor drug closely-related ten goal gene in the paraffin tissue sections sample of tumour patient, and concrete detecting step is as follows:
1, collecting sample, extracts RNA from the paraffin tissue sections of tumour patient.
Paraffin-embedded tissue is cut into 10 μm of thin slices, gets thin slice 5, use paraffin RNA to extract test kit, extract RNA.Measure after concentration through ultraviolet spectrophotometer, 4 DEG C or-20 DEG C save backup.
2, with the tumor tissues RNA extracted for template, reverse transcription becomes cDNA.
Use the mixture of RT primer, step 1 gained RNA reverse transcription is become cDNA.The component of the RT reaction solution prepared is as shown in table 2.
Table 2 RT reaction solution component list
The reaction conditions of reverse transcription is: 48 DEG C of 1min, 42 DEG C of 60min, 95 DEG C of 5min, remains on 4 DEG C until collect RT product.Be about 75min. reverse transcription product total time and can preserve the several months at-20 DEG C.
3, take cDNA as template, carry out multi-PRC reaction.
CDNA reverse transcription in step 2 obtained is template, uses pcr amplification primer, forward universal primer and reverse universal primer, amplification PCR primer.The composition of the PCR reaction solution prepared is as shown in table 3.
Table 3 PCR reaction solution component list
Reaction system after preparing is reacted on instrument at PCR and is run following program:
（1）95℃ 10min；
（2）94℃ 30sec；
（3）55℃ 30sec；
（4）72℃ 30sec；
(5) circulation of 34, (2) ~ (4) is repeated;
（6）72℃ 5min；
(7) 4 DEG C of insulations.
4, use GenomeLab GeXP genetic analyzer, carry out capillary electrophoresis, detect fluorescent signal, and carry out data analysis.
Sample-loading buffer (methane amide, SLS) 38.5 μ L is added, interior mark Marker(DNA Standard 400 in every hole of 96 hole sample panel) 0.5 μ L, multiple PCR products 1 μ L, fully add 1 mineral oil after mixing, prevent methane amide to be oxidized.
The GenomeLab dissociating buffer of about 250 μ L is added in every hole of another 96 hole damping fluid plate.
According to the operational manual of GeXP genetic analyzer, kapillary and gel are installed.Sample panel and damping fluid plate are put into machine, runs Frag-3 separable programming.Perform the GeXP analytical procedure of acquiescence, finally preserve 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 to give tacit consent to analytical standard " Default GeXP Analysis Parameters ", carry out fragment analysis.
Fragment analysis result is imported 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 each gene after homogenization.Homogenization can be eliminated by different the brought error 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, and ordinate zou expression signal is strong and weak.Corresponding to each gene amplification, the calculated by peak area at peak obtains the relative expression levels of anti-tumor drug genes involved.Fig. 1 adopts test kit of the present invention carry out RT, PCR to the RNA mixture that kinds of tumor cells is extracted and utilize 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.Result judged it is as reference with the gene expression dose of normal people.
Four, the sensitivity of test kit and specificity analyses.
Sensitivity analysis: 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 being 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, is detected as the unimodal of 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 exhaustive without the need to also giving all embodiments.And these belong to spirit institute's apparent change of extending out of the present invention or change and are still among protection scope of the present invention.
Claims (8)
1. a multiple gene detection kit relevant to anti-tumor drug, comprise the mixture of RT primer and the mixture of pcr amplification primer, the mixture of described RT primer comprises based on each RT primer of target in gene group, reference gene and reaction, the mixture of described pcr amplification primer comprises based on 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;
The forward universal primer sequence that described each pcr amplification primer sequence is held by specific primer sequence and 5 ' forms, and the reverse universal primer sequence that described each RT primer sequence is held by specific primer sequence and 5 ' forms;
Based on the specific primer sequence of the pcr amplification primer of described PTEN as shown in SEQ ID No.1, the specific primer sequence of RT primer is as shown in SEQ ID No.2;
Based on the specific primer sequence of the pcr amplification primer of described EGFR as shown in SEQ ID No.3, the specific primer sequence of RT primer is as shown in SEQ ID No.4;
Based on the specific primer sequence of the pcr amplification primer of described DPYD as shown in SEQ ID No.5, the specific primer sequence of RT primer is as shown in SEQ ID No.6;
Based on the specific primer sequence of the pcr amplification primer of described HER2 as shown in SEQ ID No.7, the specific primer sequence of RT primer is as shown in SEQ ID No.8;
Based on the specific primer sequence of the pcr amplification primer of described RRM1 as shown in SEQ ID No.9, the specific primer sequence of RT primer is as shown in SEQ ID No.10;
Based on the specific primer sequence of the pcr amplification primer of described ERCC1 as shown in SEQ ID No.11, the specific primer sequence of RT primer is as shown in SEQ ID No.12;
Based on the specific primer sequence of the pcr amplification primer of described TUBB3 as shown in SEQ ID No.13, the specific primer sequence of RT primer is as shown in SEQ ID No.14;
Based on the specific primer sequence of the pcr amplification primer of described TOP1 as shown in SEQ ID No.15, the specific primer sequence of RT primer is as shown in SEQ ID No.16;
Based on the specific primer sequence of the pcr amplification primer of described TYMS as shown in SEQ ID No.17, the specific primer sequence of RT primer is as shown in SEQ ID No.18;
Based on the specific primer sequence of the pcr amplification primer of described TOP2A as shown in SEQ ID No.19, the specific primer sequence of RT primer is as shown in SEQ ID No.20;
Based on the specific primer sequence of the pcr amplification primer of described ACTB as shown in SEQ ID No.21, the specific primer sequence of RT primer is as shown in SEQ ID No.22;
Based on the specific primer sequence of the pcr amplification primer of described GAPDH as shown in SEQ ID No.23, the specific primer sequence of RT primer is as shown in SEQ ID No.24;
Based on the specific primer sequence of the pcr amplification primer of described B2M as shown in SEQ ID No.25, the specific primer sequence of RT primer is as shown in SEQ ID No.26;
Based on the specific primer sequence of the pcr amplification primer of described KAN-r as shown in SEQ ID No.27, the specific primer sequence of RT primer is as shown in SEQ ID No.28.
2. a kind of multiple gene detection kit relevant to anti-tumor drug according to claim 1, it is characterized in that: the concentration of pcr amplification primer in reaction system based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene is 150nM ~ 250nM, the concentration of RT primer in reaction system based on PTEN gene is 10nM ~ 20nM; The concentration of RT primer in reaction system based on EGFR gene is 1800nM ~ 2200nM; The concentration of RT primer in reaction system based on DPYD, RRM1, TYMS and KAN-r gene is 450nM ~ 550nM; The concentration of RT primer in reaction system based on HER2 gene is 50nM ~ 70nM; The concentration of RT primer in reaction system based on ERCC1 and TOP1 gene is 200nM ~ 300nM; The concentration of RT primer in reaction system based on TUBB3 and TOP2A gene is 800nM ~ 1200nM; The concentration of RT primer in reaction system based on ACTB gene is 20nM ~ 40nM; The concentration of RT primer in reaction system based on GAPDH and B2M gene is 8nM ~ 12nM.
3. a kind of multiple gene detection kit relevant to anti-tumor drug according to claim 2, it is characterized in that: the concentration of pcr amplification primer in reaction system based on described PTEN, EGFR, DPYD, HER2, RRM1, ERCC1, TUBB3, TOP1, TYMS, TOP2A, ACTB, GAPDH, B2M and KAN-r gene is 200nM, the concentration of RT primer in reaction system based on PTEN gene is 15nM; The concentration of RT primer in reaction system based on EGFR gene is 2000nM; The concentration of RT primer in reaction system based on DPYD, RRM1, TYMS and KAN-r gene is 500nM; The concentration of RT primer in reaction system based on HER2 gene is 60nM; The concentration of RT primer in reaction system based on ERCC1 and TOP1 gene is 250nM; The concentration of RT primer in reaction system based on TUBB3 and TOP2A gene is 1000nM; The concentration of RT primer in reaction system based on ACTB gene is 30nM; The concentration of RT primer in reaction system based on GAPDH and B2M gene is 10nM.
4., according to a kind of relevant to the anti-tumor drug multiple gene detection kit one of claims 1 to 3 Suo Shu, it is characterized in that: the mixture also comprising 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 concentration of reverse universal primer in reaction system are 1.0 μMs ~ 1.5 μMs.
5. a kind of multiple gene detection kit relevant to anti-tumor drug according to claim 4, is characterized in that: the fluorescent mark of described forward universal primer comprises Cy5, Cy3 or FAM.
6. a kind of multiple gene detection kit relevant to anti-tumor drug according to claim 5, is characterized in that: described forward universal primer and the concentration of reverse universal primer in reaction system are 1.2 μMs.
7. according to a kind of relevant to the anti-tumor drug multiple gene detection kit one of claims 1 to 3 Suo Shu, it is characterized in that: also comprise ThermoScript II, reverse transcription buffer, KAN-r RNA, archaeal dna polymerase, PCR damping fluid, dNTP and ultrapure water.
8. according to a kind of relevant to the anti-tumor drug multiple gene detection kit one of claims 1 to 3 Suo Shu, it is characterized in that: also comprise positive reference substance, described positive reference substance is the RNA mixture extracted from several tumour cells, and described positive reference substance comprises target mRNA in described gene group, reference gene and reaction.
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| CN108504733A (en) * | 2017-11-29 | 2018-09-07 | 中山拓普基因科技有限公司 | Tumour Individual Chemotherapy medication guide gene SNP site detection combination object |
| CN108456730B (en) * | 2018-02-27 | 2021-01-05 | 海门善准生物科技有限公司 | Application of recurrence risk gene group as marker in preparation of product for evaluating recurrence risk at distant place in breast cancer molecular typing |
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