CN102312002A

CN102312002A - Kit for rapid detection of mRNA expression level of BRCA1 gene

Info

Publication number: CN102312002A
Application number: CN201110279225A
Authority: CN
Inventors: 李艳; 童永清
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-09-20
Filing date: 2011-09-20
Publication date: 2012-01-11

Abstract

The invention relates to a fluorescence quantitative PCR (Polymerase Chain Reaction) diagnostic kit for rapid detection of mRNA expression level of a BRCA1 gene. The kit comprises BRCA1gene primer, a reference gene GAPDH primer and a Taqman fluorescence probe. The BRCA1 is an important cancer suppressor gene, and proteins coded by the BRCA1 gene play an important role in DNA damage and repair. A fluorescence quantitative PCR with high sensitivity and specificity is employed to detect the mRNA expression level of BRCA1; and a detection result has substantially increased specificity and sensitivity. The kit provides a novel rapid simple gene diagnostic technique for whether a clinical malignant tumor patient should use platinum chemotherapeutics and antimicrotubular medicaments.

Description

A kind of test kit of rapid detection BRCA1 gene mRNA expression amount

Technical field

The present invention relates to biological technical field, be specifically related to a kind of test kit that utilizes fluorescent quantitative PCR technique rapid detection BRCA1 gene mRNA expression amount.

Background technology

Platinum compound is the tumor chemotherapeutic drug that the most often uses, and comprises cis-platinum, carboplatin and oxaliplatin etc., has characteristics such as anticancer spectrum is wide, effect is strong.Because platinum medicine, causes interlinkage in DNA interchain interlinkage or the chain through broad incorporation DNA, causes dna damage, thereby cause necrocytosis and realize anticancer purpose.Clinical application shows that there is significant difference between individuals in the curative effect of platinum medicine, and after platinum class cytotoxicity chemotherapeutics damaged tumour, the DNA repair mechanism of tumour cell started, and the tumour cell with higher repair ability is easy to chemotherapy resistance.BRCA1 plays an important role in DNA repairs, and is in close relations with platinum class resistance, and the patient that promptly the BRCA1 gene expression dose is low is responsive to platinum medicine, otherwise the high patient of expression level shows resistance.The height of discovering the BRCA1 gene expression dose is all relevant with morbidity, biological characteristics and the prognosis of mammary cancer, ovarian cancer and nonsmall-cell lung cancer.Reported literature is arranged, in accepting the nonsmall-cell lung cancer patient of platinum medicine chemotherapy than late period, the more negative expresser's poor prognosis of BRCA1 positive cases, the high expression level of BRCA1 is to cause tumour cell to one of drug-fast most important mechanism of platinum medicine.

Microtubule based chemotherapy medicine is to act on the cell microtubule, forms through influencing spindle body, thus a based chemotherapy medicine of inhibition cell mitogen.Anti-microtubule class medicine commonly used at present mainly contains: taxol, Docetaxel, vinealeucoblastine(VLB), vincristine(VCR) and vinorelbine etc.The anticancer spectrum of anti-microtubule class medicine is very wide, is to use antitumor drug the most widely.But in clinical application, but occur the efficient low and big problem of spinoff of treatment often, have significant difference between individuals.Vast amount of clinical shows that the mRNA expression level of BRCA1 gene is closely related in the curative effect of anti-microtubule medicine and the tumor tissues, and the patient that promptly the BRCA1 gene expression dose is high resists microtubule class medicaments insensitive, otherwise the low patient of expression level shows resistance.

The low patient who expresses of the mRNA of BRCA1 accepts lifetime behind the platinum-based chemotherapy than high expression level patient phenomenal growth, and in the patient of BRCA1 mRNA high expression level, the ratio of accepting taxol/platinum-based chemotherapy is only accepted patient's survival period of platinum-based chemotherapy.Therefore, the detection of BRCA1 genetic expression has adapted to this requirement of tumour individualized treatment, and chemotherapeutics is selected to have directive significance.And the mRNA level of fluorescence quantitative PCR detection BRCA1 is all more higher than using immunohistochemical methods detection by quantitative BRCA1 its susceptibility of protein content and specificity.

Still the test kit that does not have at present reported literature related detection BRCA1 gene mRNA expression amount.

Summary of the invention

Technical problem to be solved by this invention is to provide the test kit of quick, easy, responsive, the special detection BRCA1 gene mRNA expression amount of a kind of ability.

Test kit of the present invention utilizes fluorescent quantitative PCR technique, comprises BRCA1 gene primer, internal control gene (GAPDH) primer and Taqman fluorescent probe:

BRCA1 upstream region of gene primer sequence is: 5 '-ATGGGCCCTTCACCAACA-3 ';

BRCA1 gene downstream primer sequence is: 5 '-CACAGAAGCACCACACAGCTGTA-3 ';

Taqman fluorescent probe: 6FAM 5 '-ATCAACTGGAATGGATG-3 ' TAMRA;

GAPDH upstream region of gene primer sequence is: 5 '-ATGGAAATCCCATCACCATCTT-3 ';

GAPDH gene downstream primer sequence is: 5 '-CGCCCCACTTGATTTTGG-3 ';

Taqman fluorescent probe: 6FAM 5 '-CAGGAGCGAGATCC-3 ' TAMRA.

Described test kit comprises also and carries out the required reagent of quantitative fluorescent PCR that specifically comprise: the first chain cDNA synthetic agent, negative control and positive control, PCR reaction solution, RNA extract reagent.

The first chain cDNA synthetic agent wherein is: 25mmol/L MgCl ₂4 μ l, 10 * reversed transcriptive enzyme damping fluid, 2 μ l, 10mmol/L dNTP 2 μ l, RNA enzyme inhibitors 0.5 μ l, Oligo (dT) ₁₅0.5 μ g, AMV reversed transcriptive enzyme 15U, no RNase deionized water.

Wherein negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of the total RNA sample of BRCA1.

PCR reaction solution wherein comprises: the primer of 10 * PCR reaction mixture, 0.25pmol/ μ l (BRCA1 gene primer and internal control gene primer), the fluorescent probe of 0.3pmol/ μ l, the MgCl of 2.5～4.0mM ₂, 2U Taq enzyme, 0.2～0.4mM dNTPs, 0.2～1U UNG enzyme, 0.3～0.6mM dUTP, get the template of 1～2 μ l usually.

Detect BRCA1 gene mRNA expression amount with test kit of the present invention: at first obtain the clinical tumor tissue samples, rapid extraction is organized RNA, carries out the synthetic first chain cDNA of reverse transcription PCR; Dispose the PCR reaction solution then and carry out quantitative fluorescent PCR, in the quantitative real time PCR Instrument data analysis system, read CT value result.Calculate the CT value of BRCA1 and GAPDH gene respectively, both differences are Δ Ct value.Fluorescent quantitative PCR result adopts software analysis, and markization calculating sampled data.

The advantage and the beneficial effect of test kit of the present invention are following:

(1) sensitivity: the fluorescent PCR detection technique is that to combine round pcr, fluorescent mark technology, laser technology, digital visualization techniques be the technology of one, so its detection sensitivity is very high.

(2) special: as to use specific probe that quantitative molecular is discerned, have very high accuracy.Simultaneously, target sequence is by primer and the dual control of probe, and specificity is good, false positive is low.

(3) handy and safe: simple to operate, safety, level of automation are high, anti-pollution.Amplification and detection can detect in same pipe, need not uncap, and are difficult for polluting; Increasing simultaneously and detecting one goes on foot completion, does not need post-processed, no longer need worry radiocontamination.

(4) quick: speed is fast, high-throughput, can accomplish at 3～4 hours.

Description of drawings

Fig. 1 is the fluorescence curve figure of fluorescence real-time quantitative PCR detection BRCA1 gene standard substance, and ordinate zou is a fluorescence intensity, and X-coordinate is a cycle index.

The typical curve that Fig. 2 obtains for the fluorescence curve figure according to BRCA1 gene standard substance, ordinate zou is a cycle index, X-coordinate is the logarithmic value of fluorescence intensity.

Fig. 3 is the fluorescence curve figure of fluorescence real-time quantitative PCR detection GAPDH standard substance, and ordinate zou is a fluorescence intensity, and X-coordinate is a cycle index.

The typical curve that Fig. 4 obtains for the fluorescence curve figure according to the GAPDH standard substance, ordinate zou is a cycle index, X-coordinate is the logarithmic value of fluorescence intensity.

Embodiment

Combine embodiment and accompanying drawing at present, the present invention is further described, but enforcement of the present invention is not limited in this.

The preparation of embodiment 1. test kits of the present invention

(1) test kit of the present invention is formed as follows:

1. Trizol: rapid extraction cancerous tissue RNA;

2. the first chain cDNA synthetic agent box (RT-PCR); Comprise 25mmol/L MgCl ₂4 μ l, 10 * reversed transcriptive enzyme damping fluid, 2 μ l, 10mmol/L dNTP 2 μ l, RNA enzyme inhibitors 0.5 μ l, Oligo (dT) ₁₅0.5 μ g, AMV reversed transcriptive enzyme 15U, no RNase deionized water;

3. primer: comprise goal gene BRCA1, internal control gene GAPDH and fluorescent probe, specific as follows:

BRCA1 gene primer sequence:

BRCA1 upstream region of gene primer sequence is: 5 '-ATGGGCCCTTCACCAACA-3 '; (SEQ ID No.1)

BRCA1 gene downstream primer sequence is: 5 '-CACAGAAGCACCACACAGCTGTA-3 '; (SEQID No.2)

Taqman fluorescent probe: 6FAM 5 '-ATCAACTGGAATGGATG-3 ' TAMRA; (SEQ ID No.3)

GAPDH gene primer sequence:

GAPDH upstream region of gene primer sequence is: 5 '-ATGGAAATCCCATCACCATCTT-3 '; (SEQ IDNo.4)

GAPDH gene downstream primer sequence is: 5 '-CGCCCCACTTGATTTTGG-3 '; (SEQ IDNo.5)

Taqman fluorescent probe: 6FAM 5 '-CAGGAGCGAGATCC-3 ' TAMRA.(SEQ?ID?No.6)

Above-mentioned primer sequence, probe sequence are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

4. negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of the total RNA sample of BRCA1.

5. PCR reaction solution: the primer of 10 * PCR reaction mixture, 0.25pmol/ μ l, the probe of 0.3pmol/ μ l, the Mg of 2.5～4.0mM ²⁺, 2U UNG enzyme, 0.3～0.6mMdUTP, template, the reaction TV of getting 1～2ul usually of dNTPs, 0.2～1U of Taq enzyme, 0.2～0.4mM be generally 20 μ l (above all concentration all is meant final concentration)

The setting of pcr amplification program: first 50 ℃ of 10s normally on the lightcycler2.0 instrument, 95 ℃ of 10min, 95 ℃ of 15s then, 60 ℃ of 1min circulate 45 times.

The test kit of embodiment 2. usefulness embodiment 1 preparation detects the expression amount of BRCA1 mRNA

To detect 20 routine mammary cancer paraffin section sample organize results is example.

Testing process:

At first according to the primer and the fluorescent probe of gene order designs specificity.Obtain clinical breast cancer tissue sample, rapid extraction is organized RNA, carries out the synthetic first chain cDNA of RT-PCR; Configuration PCR reaction solution carries out quantitative fluorescent PCR.In the LightCycler data analysis system, read CT value result.Calculate the CT value of BRCA1 and GAPDH gene respectively, both differences are Δ Ct value.Fluorescent quantitative PCR result adopts software analysis, and markization calculating sampled data.

Concrete steps are following:

1. the extracting of total tissue RNA: the method extracting cancer and the total RNA of cancer beside organism that press the RNA extracting and purifying.The RNA that extracts identifies its integrity through agarose gel electrophoresis, through purity and the concentration of ultraviolet spectrophotometer mensuration 260nm and 280nm OD value calculating RNA, regulates extractive RNA to same concentrations with the water that 0.1% (mass volume ratio) DEPC handles.

2. cDNA is synthesized in reverse transcription: get the above-mentioned RNA liquid of 2 μ l and add 25mmol/L MgCl subsequently at 70 ℃ of insulation 10min ₂4 μ l, 10 * reversed transcriptive enzyme damping fluid, 2 μ l, 10mmol/L dNTP 2 μ l, RNA enzyme inhibitors 0.5 μ l, Oligo (dT) ₁₅0.5 μ g, AMV reversed transcriptive enzyme 1.5U adds DEPC treating water to TV 20 μ l, in 42 ℃ of insulation 15min, carries out reverse transcription reaction, synthetic cDNA first chain.

3. after reaction finishes, be heated to 99 ℃ of 5min with the deactivation reversed transcriptive enzyme.Add 20 μ l sterilized water mixings and put refrigerator-20 ℃ preservation.

4. fluorescent quantitative PCR: the PCR reaction system is 20 μ l: contain 2X PCR reaction mixture 10.0 μ l, BRCA1 primer concentration 0.2 μ mol/L, concentration and probe concentration 0.3 μ mol/L, cDNA1.0 μ l, ultrapure water polishing.On the lightcycler quantitative real time PCR Instrument, react: amplification condition: 95 ℃ of preparatory sex change of 10min, 95 ℃ of 15s, 45 circulations of 60 ℃ of 1min amplifications, instrument is collected fluorescent signal automatically.

5. data collection process and analysis: real-time fluorescence quantitative PCR gained data are calculated; The Ct value of analysis purposes gene (Fig. 1, Fig. 2) and house-keeping gene (Fig. 3, Fig. 4); Calculate goal gene and carry out statistical study again after with respect to the relative expression quantity of house-keeping gene (GAPDH); Is high expression level with ratio greater than 1.50, and ratio is expressed for low less than 0.50:

Sample	The BRCA1 template	The GAPDH template	BRCA1/GAPDH
				Mammary cancer	318278725	254365787	1.25
Mammary cancer	243568675	356423448	0.68
				Mammary cancer	87967548	354567645	0.25
Mammary cancer	235876547	289756856	0.81
				Mammary cancer	335795436	202458540	1.66
Mammary cancer	349898538	193235657	1.81
				Mammary cancer	302568963	201468454	1.50
Mammary cancer	267598466	255787518	1.05
				Mammary cancer	336764684	216451513	1.56
Mammary cancer	321568975	209653300	1.53
				Mammary cancer	268403709	298546190	0.90
Mammary cancer	277557900	327650194	0.85
				Mammary cancer	167954681	356406153	0.47
Mammary cancer	348569043	206487947	1.69
				Mammary cancer	317598460	210215741	1.51
Mammary cancer	253764385	286631489	0.89
				Mammary cancer	97850645	318654321	0.31
Mammary cancer	350421709	218546156	1.60
				Mammary cancer	298157590	306671049	0.97
Mammary cancer	189649078	328565890	0.58

(2) the test kit detectivity is estimated:

According to instance 1; Adopt test kit of the present invention to detect through patient's sample that immunohistochemical method detects the BRCA1 protein expression 45 examples; The detectivity of the two is contrasted; This test kit susceptibility, specificity and sensitivity and immunohistochemical method compare, and this test kit is more accurate, meet the practical requirement of present clinic diagnosis fully:

Wherein:

1. specificity: 93.3%;

2. sensitivity: 100.0%;

3. positive predictive value: positive predictive value reaches 96.8%;

4. negative predictive value: negative predictive value reaches 100.0%;

5. repeated: repeatedly the repeated experiments result is consistent;

6. consuming time: as to be about 3～4h the detection time of a clinical samples, weak point consuming time.

Above-mentioned experiment can be explained; Adopt susceptibility and all higher fluorescence quantitative PCR detection BRCA1mRNA level of specificity; The specificity and the susceptibility of its detected result are significantly increased, and this test kit is that the chemotherapy and the prognosis of clinical tumor provides a kind of brand-new gene diagnosis fast and accurately technology.

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Claims

1. the test kit of a rapid detection BRCA1 gene mRAN expression amount comprises the BRCA1 gene primer, internal control gene GAPDH primer and Taqman fluorescent probe:

BRCA1 upstream region of gene primer sequence is: 5 '-ATGGGCCCTTCACCAACA-3 ';

BRCA1 gene downstream primer sequence is: 5 '-CACAGAAGCACCACACAGCTGTA-3 ';

Taqman fluorescent probe: 6FAM 5 '-ATCAACTGGAATGGATG-3 ' TAMRA;

GAPDH gene downstream primer sequence is: 5 '-CGCCCCACTTGATTTTGG-3 ';

Taqman fluorescent probe: 6FAM 5 '-CAGGAGCGAGATCC-3 ' TAMRA;

Also comprise: the first chain cDNA synthetic agent, negative control and positive control, PCR reaction solution, RNA extract reagent.

2. test kit according to claim 1 is characterized in that, the first chain cDNA synthetic agent wherein is: 25 mmol/L MgC1 ₂4 μ l, 10 * reversed transcriptive enzyme damping fluid 2ul, 10mmol/L dNTP 2 μ l, RNA enzyme inhibitors 0.5 μ l, Oligo (dT) ₁₅0.5 μ g, AMV reversed transcriptive enzyme 15U, no RNase deionized water.

3. test kit according to claim 1 and 2 is characterized in that, wherein negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of the total RNA sample of BRCA1.

4. test kit according to claim 1 and 2 is characterized in that, PCR reaction solution wherein comprises: the primer of 10 * PCR reaction mixture, 0.25pmol/ μ l, the fluorescent probe of 0.3pmol/ μ l, the MgCl of 2.5 ~ 4.0mM ₂, the Taq enzyme of 2U, the dNTPs of 0.2 ~ 0.4mM, the UNG enzyme of 0.2 ~ 1U, 0.3 ~ 0.6mM dUTP.