CN104131103B - AMZ1 gene is preparing the purposes in diagnostic kit - Google Patents

AMZ1 gene is preparing the purposes in diagnostic kit Download PDF

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CN104131103B
CN104131103B CN201410389636.XA CN201410389636A CN104131103B CN 104131103 B CN104131103 B CN 104131103B CN 201410389636 A CN201410389636 A CN 201410389636A CN 104131103 B CN104131103 B CN 104131103B
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amz1
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董伟伟
屈雪玲
王硕
李小梅
赵慧霞
肖文华
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First Affiliated Hospital Chinese PLA General Hospital
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Abstract

The invention discloses the purposes of AMZ1 gene in the test kit of preparation diagnosing colon cancer.Does the present invention utilize NCBI? GEO data retrieval includes satisfactory 5 cover colon cancer cases and normal population genome mrna expression chip data in research range, by carrying out Meta analysis to said chip data, filtering out AMZ1 gene is significant difference gene, compare healthy tissues, AMZ1 gene down-regulated expression in colon cancer tissue, and use the above-mentioned conclusion of fluorescence real-time quantitative PCR method validation.Accordingly, the invention also discloses a kind of test kit of diagnosing colon cancer and utilize the method for this test kit diagnosing colon cancer.One aspect of the present invention provides new thinking, on the other hand for early diagnosis colorectal carcinoma provides more sensitive diagnostic kit for studying the molecule mechanism of colorectal carcinoma.

Description

AMZ1 gene is preparing the purposes in diagnostic kit
Technical field
The invention belongs to biomedicine field, relate to a kind of diagnosis of colon cancer test kit and AMZ1 gene is preparing the application in diagnosis of colon cancer test kit.
Background technology
To apply the earliest in transcript profile research and the most widely for biochip technology, first from detected sample, RNA is extracted, and utilize fluorescently-labeled Nucleotide that its reverse transcription is become cDNA, nucleotide sequence through mark can with the probe hybridization on gene chip specific site, after testing hybridization signal and obtain cellular gene expression information.Biochip technology has become a highly stable believable experimental technique, and a large amount of transcript profile data announced at present mainly utilize biochip technology to produce.
Colorectal carcinoma is the common cancer of masculinity and femininity the 3rd, points out 2012 annual U.S. colorectal carcinoma new cases 103170 in american cancer statistical reports in 2013.And in China, along with the change of mode of life and food habits in recent years, colorectal carcinoma morbidity and death rise all fast, according to statistical result showed in 2011, colorectal carcinoma occupies China's Cancer Mortality the 3rd, be the 4th and cause dead malignant tumour, M & M, all close to contemporaneously world standard, becomes the public health problem of serious threat crowd life and health.The pathogenesis of colorectal carcinoma is illustrated not yet completely, but Study of Etiology shows, most colorectal carcinoma be a multi-step, multistage, environmental factors and the coefficient complex process of inherited genetic factors.Its generation development relates to the change of multiple gene, shows as collaborative accumulation and the interaction of polygene multi-step.In recent years, the technology such as applying gene chip and high flux screening had carried out more research to colorectal carcinoma gene expression profile both at home and abroad.But the difference of different experiment porchs and sample causes analytical results to there is a lot of discordance.
Meta analyzes that the result can delivering correlative study report to same problem institute is collected, statistical integration, to the result that acquisition is more accurate or more.This is analyzed and can produce a lot of significant difference expression gene, can avoid the incorrectness that single research brings.Meta analysis is the powerful tool of Recognition Different expressing gene in multiple array experiment research.In addition, analyze the differential gene that can identify single research and can not identify with Meta, and effectively can reduce the false positive of one single chip data.
The present invention is by carrying out Meta analysis to the colorectal carcinoma transcript profile expression data delivered, filter out and affect the key gene that development occurs colorectal carcinoma, and use the expression in fluorescence real-time quantitative PCR (QPCR) detection validation clinical sample, the present invention excavates the effect of these genes in colorectal carcinoma by information biology means, thus the pathogeny of colorectal carcinoma is probed into, for its Diagnosis and Treat provides certain Research foundation.
Summary of the invention
The present invention, by carrying out Meta analysis and QPCR checking to the colorectal carcinoma transcript profile expression data delivered, finds that the expression of AMZ1 gene in colon cancer tissue there are differences with expression in the normal tissue.Compared with healthy tissues, AMZ1 gene down-regulated expression in colon cancer tissue, by detecting the expression of AMZ1 gene transcription level, can judge whether experimenter suffers from colorectal carcinoma.
AMZ1 gene is the object of the present invention is to provide to prepare the purposes in diagnosis of colon cancer test kit.Described diagnostic kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase AMZ1 gene and house-keeping gene.SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
In technique scheme, the prior art that PCR damping fluid is known to the skilled person, preferably, PCR damping fluid comprises: 25mMKCl, 2.5mMMgCl 2, 200mM (NH 4) 2sO 4.
Primer pair is that those skilled in the art can design according to the conventional design principle of design of primers.In preferred embodiment, the forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The preferred GAPDH of house-keeping gene, the forward primer sequence of this gene that increases is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
Preferred reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mMPH8.3,375mM, 15mM 2, the DTT of 50mM.
The RNA enzyme inhibitors that RNA enzyme inhibitors can select this area conventional, is preferably the recombinant protein enzyme of the Noncompetition inhibition RNA enzyme of escherichia coli expression.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises RNA and extracts reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
Diagnostic kit of the present invention is stored in-20 DEG C, reduces multigelation as far as possible.
Another object of the present invention is the test kit providing a kind of diagnosing colon cancer.Described diagnostic kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase AMZ1 gene and house-keeping gene.Described SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
In technique scheme, the prior art that PCR damping fluid is known to the skilled person, preferably, PCR damping fluid comprises: 25mMKCl, 2.5mMMgCl 2, 200mM (NH 4) 2sO 4.
Primer pair is that those skilled in the art can design according to the conventional design principle of design of primers; In preferred embodiment, the forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The preferred GAPDH of house-keeping gene, the forward primer sequence of this gene that increases is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
Preferred reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mMPH8.3,375mM, 15mM 2, the DTT of 50mM.
The RNA enzyme inhibitors that RNA enzyme inhibitors can select this area conventional, is preferably the recombinant protein enzyme of the Noncompetition inhibition RNA enzyme of escherichia coli expression.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises RNA and extracts reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
Present invention also offers the method for diagnosing colon cancer, described method comprises:
(1) utilize RNA to extract reagent and extract sample total serum IgE;
(2) the RNA reverse transcription that step (1) obtains is become cDNA;
(3) on fluorescence real-time quantitative PCR instrument, AMZ1 gene and house-keeping gene are carried out augmentation detection;
(4) by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification;
(5) AMZ1 down regulation of gene expression, shows that research object is colorectal cancer patients.
The specific implementation method of step (1) is: frozen in liquid nitrogen after collecting sample, organizing the mortar putting into precooling to grind, after tissue samples is powdered after taking-up:
1. Trizol is added, room temperature preservation 5min;
2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min;
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note the proteic substance be not drawn onto between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, fully put upside down mixing, be placed in 10min on ice;
4. 12000rpm carefully discards supernatant liquor at a high speed after 15min, adds 75%DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate in the ratio of 1ml/mlTrizol, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C;
5. discard ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, adds DEPC treated water dissolution precipitation;
6. RNA purity and concentration is measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.
The specific implementation method of step (2) is: carry out reverse transcription synthesis cDNA with RT Buffer to 1 μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water in PCR pipe, 5 × RT Buffer, 10mmol/LdNTP, 0.1mmol/lDTT, 30 μm of mol/lOligodT, 200U/ μ lM-MLVRT, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
The specific implementation method of step (3) is: adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBRGreen polymerase chain reaction system 12.5 μ l, forward primer (5 μMs/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l; The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ', and operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
Advantage of the present invention is with beneficial effect: it is relevant to colorectal carcinoma that (1) the present invention makes public for the first time AMZ1 gene, and AMZ1 gene is expected to the molecular marker becoming diagnosing colon cancer, and provides new thinking for studying the molecule mechanism of colorectal carcinoma.(2) utilize the existence of the mode diagnosing colon cancer of gene expression detection whether method sensitiveer, be conducive to the diagnosis that disease is early stage.
Accompanying drawing illustrates:
Fig. 1 represents that fluorescence real-time quantitative PCR is determined at the relative expression quantity of AMZ1 gene mRNA in healthy tissues and colon cancer tissue.
Concrete embodiment:
Further illustrate the present invention below in conjunction with specific embodiment, embodiments of the invention only for explaining the present invention, and do not mean that and limit the scope of the invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1 screens the gene relevant to colorectal carcinoma
1.1NCBIGEO (GeneExpressionOmnibus) data retrieval
GEO (GeneExpressionOmnibus) database is by NCBI (US National Biotechnology Information center) development and maintenance, GEO database is as the database of maximum gene expression data, this database is based on chip data, in addition the data of some non-chip types are also comprised as SAGE (serial analysis of gene expression) data, SARST (rrna sequence label is analyzed continuously) data, MS (mass spectrum) data, proteome data and high-flux sequence data of new generation (MPSS, extensive parallel sequencing technology) etc.
A. search key: (" colorectalcancer " [MeSHTerms] OR " colorectalcancer "
[AllFields])AND"Homosapiens"[porgn]
B. the screening sample strategy in research:
To be " expressionprofilingbyarray " data set of meeting following standard will include in our research restriction research type: 1. selected data collection must be the expression mRNA transcript profile data of full-length genome, and is all RNA-seq data set; 2. these data come from biopsy or the cultured cells of large bowel cancer case group and control group; 3. this research is all considered through stdn or raw data set; Finally, 5 cover RNA-seq data sets are had to include in our research (table 1).
Table 18 overlaps the basic condition of colorectal carcinoma full-length genome data set
1.2 colorectal carcinoma microarray data Meta analytical resultss
After background correction and stdn being carried out to raw data by transcript profile data analysis software, utilize microarray significance component software (significanceanalysisofmicroarray, SAM) to 5 sets of data collection standardizations, screening difference expression gene, false positive rate (falsediscoveryrate is set in analytic process, FDR) be≤0.01, filter out 883 difference expression genes altogether.
Embodiment 2QPCR verifies the relation of candidate gene and colorectal carcinoma
Based on the result analyzed colorectal carcinoma microarray data Meta, according to the size of Pvalue, we select AMZ1 gene (Meta analyzes display compared with healthy tissues, AMZ1 gene down-regulated expression in colon cancer tissue) to verify.Collect 50 colon cancer tissue samples, collect 50 healthy tissues samples simultaneously, adopt fluorescence real-time quantitative PCR to carry out classical molecular biology experiment checking (qRT-PCR), concrete operation step is as follows:
(1) RNA extracts
Frozen in liquid nitrogen after collecting sample, organizing the mortar putting into precooling to grind, after tissue samples is powdered after taking-up:
1. Trizol is added, room temperature preservation 5min;
2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min;
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note the proteic substance be not drawn onto between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, fully put upside down mixing, be placed in 10min on ice;
4. 12000rpm carefully discards supernatant liquor at a high speed after 15min, adds 75%DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate in the ratio of 1ml/mlTrizol, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C;
5. discard ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, adds DEPC treated water dissolution precipitation;
6. RNA purity and concentration is measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.
(2) reverse transcription
With RT Buffer, reverse transcription synthesis cDNA is carried out to 1 μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water in PCR pipe, 5 × RT Buffer, 10mmol/LdNTP, 0.1mmol/lDTT, 30 μm of mol/lOligodT, 200U/ μ lM-MLV, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
(3) QPCR amplification inspection
Adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBRGreen polymerase chain reaction system 12.5 μ l, forward primer (5 μMs/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l; The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ', and operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification, result as shown in Figure 1, compared with healthy tissues, the down-regulated expression of AMZ1 gene in colon cancer tissue, consistent with Meta analytical results.
The preparation of embodiment 3 diagnosis of colon cancer test kit
According to the dependency of AMZ1 gene and colorectal carcinoma, diagnosing colon cancer can be carried out by the expression detecting AMZ1 gene whether to occur, the invention provides a kind of test kit carrying out diagnosing colon cancer based on detection AMZ1 genetic expression accordingly, the component in this diagnostic kit is as follows: SYBRGreen polymerase chain reaction system; The primer pair of amplification AMZ1 gene and GAPDH gene.The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.PCR buffer components is: 25mMKCL, 2.5mMMgCL 2, 200mM (NH 4) 2sO 4.
The preparation of embodiment 4 diagnosis of colon cancer test kit
According to the dependency of AMZ1 gene and colorectal carcinoma, diagnosing colon cancer can be carried out by the expression detecting AMZ1 gene whether to occur, the invention provides a kind of test kit carrying out diagnosing colon cancer based on detection AMZ1 genetic expression accordingly, the component in this diagnostic kit is as follows: primer pair, the M-MLV reverse transcription system of SYBRGreen polymerase chain reaction system, amplification AMZ1 gene and GAPDH gene.The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.PCR buffer composition is: 25mMKCL, 2.5mMMgCL 2, 200mM (NH 4) 2sO 4.M-MLV reverse transcription system component is: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Reverse transcription reaction fluid component is: 250mMTris-HCL (PH8.3), 375mMKCL, 15mMMgCL 2, 50mM DTT.RNA enzyme inhibitors is the recombinant protein enzyme of the Noncompetition inhibition RNA enzyme of escherichia coli expression.
The preparation of embodiment 5 diagnosis of colon cancer test kit
According to the dependency of AMZ1 gene and colorectal carcinoma, diagnosing colon cancer can be carried out by the expression detecting AMZ1 gene whether to occur, the invention provides a kind of test kit carrying out diagnosing colon cancer based on detection AMZ1 genetic expression accordingly, the component in test kit is as follows: primer pair, the RNA of SYBRGreen polymerase chain reaction system, amplification AMZ1 gene and GAPDH gene extract reagent.The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.PCR buffer composition is: 25mMKCL, 2.5mMMgCL 2, 200mM (NH 4) 2sO 4.RNA extracts pack containing Trizol, chloroform, Virahol, 75% ethanol.
The preparation of embodiment 6 diagnosis of colon cancer test kit
According to the dependency of AMZ1 gene and colorectal carcinoma, diagnosing colon cancer can be carried out by the expression detecting AMZ1 gene whether to occur, the invention provides a kind of test kit carrying out diagnosing colon cancer based on detection AMZ1 genetic expression accordingly, reagent constituents is as follows: the primer pair of SYBRGreen polymerase chain reaction system, amplification AMZ1 gene and GAPDH gene, M-MLV reverse transcription system, RNA extract reagent.The forward primer sequence of amplification AMZ1 gene is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.SYBRGreen polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.PCR buffer composition is: 25mMKCL, 2.5mMMgCL 2, 200mM (NH 4) 2sO 4.M-MLV reverse transcription system component is: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Reverse transcription reaction fluid component is: the MgCL of the Tris-HCL (PH8.3) of 250mM, KCL, 15mM of 375mM 2, the DTT of 50mM.RNA enzyme inhibitors is the recombinant protein enzyme of the Noncompetition inhibition RNA enzyme of escherichia coli expression.RNA extracts pack containing Trizol, chloroform, Virahol, 75% ethanol.

Claims (5)

1. detecting the reagent of AMZ1 gene preparing the purposes in diagnosis of colon cancer test kit, it is characterized in that, described diagnostic kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase AMZ1 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
2. purposes according to claim 1, it is characterized in that, the sequence of the primer pair of described amplification AMZ1 gene is as follows: forward primer sequence is 5 '-AAGAACAACAAGCCAGGGGACG-3 ', and reverse primer sequences is 5 '-CTGGAAGGAACTTGCTGAAGGTGA-3 '; Described house-keeping gene is GAPDH, and the forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
3. purposes according to claim 1, is characterized in that, described PCR damping fluid comprises: 25mMKCl, 2.5mMMgCl 2, 200mM (NH 4) 2sO 4.
4. the purposes according to any one of claim 1-3, is characterized in that, described diagnostic kit also comprises M-MLV reverse transcription system or RNA extracts reagent; Described reverse transcription system comprises T and repeats oligonucleotide OligodT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs; Described RNA extracts pack containing Trizol, chloroform, Virahol, 75% ethanol.
5. purposes according to claim 4, is characterized in that, described reverse transcription reaction liquid comprises: the MgCl of KCl, 15mM of Tris-HCl, 375mM of 250mMpH8.3 2, 50mM DTT.
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