CN102994627A

CN102994627A - Detection method of individual guidance of drug for tumor

Info

Publication number: CN102994627A
Application number: CN2012102065834A
Authority: CN
Inventors: 吴勇; 李令栋; 南丽; 颜进; 王晴晴
Original assignee: HEALTH BIOLOGICAL MEDICINE CO Ltd
Current assignee: NINGBO HEALTH GENE TECHNOLOGIES CO., LTD.
Priority date: 2012-06-21
Filing date: 2012-06-21
Publication date: 2013-03-27
Anticipated expiration: 2032-06-21
Also published as: CN102994627B

Abstract

The invention discloses a detection method of individual guidance of a drug for tumor. The method comprises the following steps of: (1) collecting a patient sample and extracting nucleic acid; (2) performing reverse transcription by taking the nucleic acid of the patient as a template; (3) performing PCR (polymerase chain reaction) by taking the reverse transcription product as a template; and (4) separating the sample by a capillary electrophoresis method. The detection method of individual guidance of the drug for tumor disclosed by the invention has the advantages of high sensitivity, good repeatability, high accuracy, high flexibility and low cost.

Description

The detection method that a kind of tumour drug usage individuation instructs

Technical field

The present invention relates to a kind of detection method, the detection method that especially a kind of highly sensitive, good reproducibility, the tumour drug usage individuation that accuracy is strong, handiness is strong, cost is low instruct.

Background technology

That uses at present often has real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., all is to detect for the specific target gene.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of fluorescent quantitative PCR technique is that susceptibility is high, but and accurate quantification, to EGFR, BRAF has obtained good result in the detection of KRAS etc.Utilize special primer that the sudden change target sequence is carried out precisely pcr amplification amplification of height, and the detection that utilizes a kind of novel probe in the real-time fluorescence quantitative PCR platform is realized sample DNA, to suddenly change, very high specificity and susceptibility had.Wherein SFDA " medicine equipment registration certificate " and the CE of European Union authentication have been obtained at the most important EGFR of tumour individuation molecular diagnosis field, KRAS, three kinds of detection in Gene Mutation reagent of BRAF product at present.The shortcoming of quantitative fluorescent PCR: flux is low, once can only detect a gene, detects multiple associated gene mutation such as needs, SNP, and expression just needs multiple pc R instrument to work simultaneously, and efficient is low, and the cycle is long; Cost is relatively high: because once detecting a gene locus, when a sample needs to detect the kinds of tumors genes involved simultaneously, must detect one by one, cost increases relatively; Be not suitable for the pathological tissue that detects after the fixedly embedding, but the majority that the patient can supply is the tissue after fixing; Can only set a reference gene (house-keeping gene), but the house-keeping gene of tumor tissues is different from healthy tissues, needs one group of house-keeping gene to make confidential reference items; Can only the maximum three kinds of genes of synchronous detection, but individualized treatment needs tens kinds of synchronous detection even more gene.

Summary of the invention

The purpose of this invention is to provide the detection method that a kind of highly sensitive, good reproducibility, accuracy is strong, handiness is strong, cost is low tumour drug usage individuation instruct.

The present invention adopts following technical scheme:

The detection method that a kind of tumour drug usage individuation instructs comprises the steps: that (1) gathers patient's sample, and extracts nucleic acid; (2) carry out reverse transcription take patient's nucleic acid as template; (3) carry out the PCR reaction take reverse transcription product as template; (4) with the method sample separation of electrocapillary phoresis.

Preferably, the patient's sample in the described step (1) comprises fresh, freezing tumor tissues etc.

Preferably, described step (2) comprises the substep of hatching at a certain temperature in proportion behind sample panel adding reagent and sample and mixing.

Preferably, the incubation temperature in the described step (2) is respectively 48 ℃, 42 ℃, 95 ℃, 4 ℃.

Preferably, described step (3) comprises the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

Preferably, the thermal cycling temperature in the substep of described step (3) is respectively 95 ℃, 94 ℃, 55 ℃, 70 ℃, 4 ℃.

Beneficial effect of the present invention is:

1. highly sensitive, good reproducibility: adopt laser induced fluorescence(LIF)-PMT, have hypersensitivity.

2. accuracy is strong: adopt capillary electrophoresis that the PCR product is carried out separation detection, non-specific amplification product, primer dimer and specific amplification products can be separated, at utmost reduce false positive.

3. high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (such as 192 patient's samples, 11 genes involveds of each sample detection), present method can disposablely provide accurate report, avoids undetected.

4. accurate quantification: but the accurate quantification transgenation, and SNP expresses copy number.

5. handiness is strong: can adjust according to demand at any time the target gene of detection, for example newfound target site can design Auele Specific Primer immediately, puts it in " the multiple gene test scheme of tumor individual therapy medication guide " and goes.

6. cost is low: the testing cost Shao Yu $50 of each sample is beneficial to large-scale promotion.

Embodiment

The below is described in further detail the present invention according to embodiment.The present invention has founded the multiple gene test scheme of 11 kinds of kinds of tumor individualized treatments of a kind of synchronous detection genes involved.Detecting step: gather patient's sample (fresh, frozen tissue etc.), extract nucleic acid, carry out reverse transcription and PCR reaction take patient's nucleic acid as template, finally use electrocapillary phoresis method sample separation.

1. the gene and the content that detect comprise (table 1).

2. set up the reliably contrast (table 1) of sample quality and reaction process

A) the contrast confidential reference items of people RNA integrity: guarantee in checkout procedure false negative is avoided in the judgement of sample quality.

B) normal reaction contrast confidential reference items: monitoring PCR reaction efficiency, avoid false negative.

Table 1:

3. the design of primers of the multiple gene test of tumor individual therapy (seeing Table 2 nucleotide sequences)

Table 2

Concrete steps are as follows:

1. produce " the multiple gene detecting kit of tumor individual therapy ", comprise in the test kit:

1) reverse transcription damping fluid;

2) reverse transcription primer pipe (RT primer mix);

3) ThermoScript II;

4) PCR damping fluid;

5) PCR primer pipe (PCR primer mix)

6) fluorescent marker;

7) 25mM magnesium chloride (MgCl ₂);

8) archaeal dna polymerase (Taq DNAPolymerase);

9) positive control (Positive Control).

2. gather patient's sample (fresh, freezing tumor tissues etc.), and extract nucleic acid

3. carry out reverse transcription (RT) take patient's nucleic acid as template:

1) add reagent and sample (RT plate) in following ratio in 96 hole sample panel:

Annotate: positive control: 1 μ L/ reaction

2) hatch by following temperature behind the mixing:

Step	Temperature	Time
			1	48℃	1 minute
2	42℃	60 minutes
			3	95℃	5 minutes
4	4℃	Continue: until collect the RT product

4. carry out the PCR reaction take reverse transcription product as template

1) add reagent and sample (PCR plate) in following ratio in 96 hole sample panel:

The PCR reaction reagent	Amount/hole
		The PCR damping fluid, 10X	2μL
25mM MgCl2	4μL
		The PCR primer	2μL
CMO1	2μL
		Archaeal dna polymerase	1.4μL
The RT product	8.6μL
		Total	20μL

2) carry out thermal cycle reaction by following temperature behind the mixing:

5. electrocapillary phoresis sample separation

1) preparation GeXP sample:

The GeXP sample	Amount/hole
		The SLS sample solution	38.75μL
DNA size criteria 400	0.5μL
		The PCR product	1μL
Total	40μL
		Mineral oil	1

Annotate: PCR product amount can be 0.1-1 μ L, or water dilutes rear loading on demand in advance

2) prepare parting liquid: about 220 μ L parting liquids are added in the hole of proper number on the 96 hole parting liquid plates

3) electrocapillary phoresis sample separation.

6. interpretation of result.

Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation and changes.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention amplifies out or change still are in protection scope of the present invention.

Claims

1. the detection method that the tumour drug usage individuation instructs comprises the steps:

(1) gathers patient's sample, and extract nucleic acid;

(2) carry out reverse transcription take patient's nucleic acid as template;

(3) carry out the PCR reaction take reverse transcription product as template;

(4) with the method sample separation of electrocapillary phoresis.

2. the detection method that instructs of a kind of tumour drug usage individuation according to claim 1, the patient's sample in the described step (1) comprises fresh, freezing tumor tissues.

3. the detection method that instructs of a kind of tumour drug usage individuation according to claim 1, described step (2) comprise in proportion and add the substep of hatching at a certain temperature behind reagent and sample and the mixing in sample panel.

4. the detection method that instructs of a kind of tumour drug usage individuation according to claim 3, the incubation temperature in the described step (2) is respectively 48 ℃, 42 ℃, 95 ℃, 4 ℃.

5. the detection method that instructs of a kind of tumour drug usage individuation according to claim 1, described step (3) comprises the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

6. the detection method that instructs of a kind of tumour drug usage individuation according to claim 5, the thermal cycling temperature in the substep of described step (3) is respectively 95 ℃, 94 ℃, 55 ℃, 70 ℃, 4 ℃.