CN109722476A - For detecting primer, probe and the kit of EGFR gene 2237-2257 mutation - Google Patents

Abstract

The invention discloses a kind of for detecting primer, probe and the kit of EGFR gene 2237-2257 mutation.The primer is as shown in SEQ ID No.1-SEQ ID No.2, and the probe is as shown in SEQ ID No.3.Real-time fluorescence PCR is carried out using kit of the invention, can detecte the EGFR gene 2237-2257 mutation for replacing with TGT.

Description

For detecting primer, probe and the kit of EGFR gene 2237-2257 mutation

Technical field

The present invention relates to field of biotechnology, specifically, being related to a kind of primer for detecting EGFR genetic mutation, visiting Needle and related kit.

Background technique

Malignant tumour is in rise year by year trend in Chinese disease incidence in recent years, and newly-increased patient is up to more than 1,700,000 people every year.Lung Cancer is one of most common malignant tumour in world wide, wherein 80%~85% is non-small cell lung cancer (nonsmall cell Lungcancer, NSCLC).In China, lung cancer has become first cause of the death of malignant tumour, 5 years survival rates about 15%.With swollen The development of tumor molecular biology, targeted therapy receive much attention, and selecting suitable patient to carry out targeted therapy is that it is successfully crucial, EGF-R ELISA (epidermal growth factor receptor, EGFR) is the important target of current oncotherapy Point.

EGFR gene is located at No. 7 the short arm of a chromosome (7p12) of people, is about 118kb, is made of 28 exons.It is transcribed The mRNA of formation is about 5.6kb, and coding molecular weight is the cross-cell membrane glycoprotein of 170kD, and intracellular region has tyrosine kinase (tyrosine kinase, TK) activity is responsible for for extracellular signal being transferred to intracellular.Abnormal EGFR activation can promote tumour The proliferation of cell, migration, differentiation, angiogenesis, and it is able to suppress the apoptosis of tumour cell.

EGFR genetic mutation occurs mainly on preceding 4 exons in the region TK intracellular (18~21 exon), studies table Bright: the patients with lung cancer that 18,19,21 exon of EGFR gene mutates takes tyrosine kinase inhibitor (tyrosine Kinase inhibitors, TKIs) class drug curative effect it is preferable, 20 exons occur mutation often have with TKIs drug resistance It closes^[1-4].In lung cancer patients in China, the mutation rate of EGFR is about 30%~50%, wherein the point mutation occurred on 18 exons is about 5% or so of EGFR mutation type is accounted for, a variety of deletion mutations occurred on 19 exons account for about 45% left side of EGFR mutation type The right side, L858R, L861Q point mutation occurred on 21 exons account for about the 40%~45% of EGFR mutation type, send out on 20 exons Raw T790M point mutation accounts for about 5% or so of EGFR mutation type^[5-6]。

Sanger PCR sequencing PCR is the main method of EGFR genetic mutation detection at present.However, the sensitivity of this method is low, meeting Lead to missing inspection and the generation of false negative, and detection time is long, is unable to satisfy the actual demand of clinical detection.Clinically compel to be essential Develop a kind of highly sensitive, quick EGFR genetic mutation detection technique.

The present inventor has found have with gastrointestinal stromal tumor in the routine testing of gastrointestinal stromal tumor (GIST) The new mutation site positioned at EGFR gene closed, i.e. 2237-2257 Substitution.The present inventor opens for the mutation A kind of quick, highly sensitive, easy to operate detection kit and relevant detection method have been sent out, it is pre- to can be used for GIST chemotherapy Afterwards.

Summary of the invention

One of the objects of the present invention is to provide a kind of for detecting the primer and probe of EGFR genetic mutation, the mutation It is 2237-2257 and replaces with TGT (base mutation: 2237-2257 > TGT, protein mutation: E746_P753 > VS).The present invention Primer and probe include following sequence:

Forward primer (F): CGTCGCTATC AAGGTGTCG (SEQ ID No:1)

Reverse primer (R): CCTGAGGTTC AGAGCCATG (SEQ ID No:2)

Probe (PB): CCCACACAGC AAAGCAGAAA CTCAC (SEQ ID No:3)

Another aspect of the present invention provides a kind of for detecting the detection kit of EGFR gene 2237-2257 mutation, institute Stating kit includes probe shown in primer shown in SEQ ID No.1-SEQ ID No.2 and SEQ ID No.3.

According to used PCR method, kit of the invention can also include reference gene and internal control gene.

Another aspect of the present invention provides a kind of method of acquisition related Ct value with EGFR gene 2237-2257 mutation, Itself the following steps are included:

(1) extract sample in genomic DNA, detection sample include fresh pathological tissue, paraffin-embedded tissue, whole blood or Blood plasma etc.；

(2) DNA is expanded with kit of the invention；

(3) the FAM fluorescence intensity for detecting reaction system obtains FAM and reaches required cycle-index when the threshold value of setting, That is Ct value.Using the Ct value as negative, Positive judgement standards.

Present invention employs specific primers and probe technique, can be with specific detection EGFR genetic mutation.The method spirit Sensitivity height, high specificity, detection speed are fast.

Detailed description of the invention

Fig. 1 is the PCR figure for detecting negative sample (wild type).

Fig. 2 is the PCR figure of detection mutation positive sample.

Specific embodiment

Combined with specific embodiments below, the present invention is further described.It should be understood that these embodiments are used merely to explain this hair It is bright rather than limit the scope of the invention.Test method without specific conditions in embodiment, according to those skilled in the art Normal condition known to member, or the condition suggested according to manufacturer.

One, principle

Specific primer of the invention and probe with DNA profiling in conjunction with after, Taq archaeal dna polymerase is with deoxynucleotide (dNTP) it is substrate, the mutated genes of reference gene (Internal Reference, IR) and EGFR gene is carried out external Amplification.Detection uses Fluorescence PCR assay, hydrolyzes release fluorescence by specific probe, the progress of monitoring PCR reaction determines EGFR genetic mutation situation.

Reference gene detection architecture has been separately provided in kit of the invention.Reference gene is to be different from EGFR gene to be checked House-keeping gene.By the amplification situation (channel FAM) of detection reference gene, it can analyze whether DNA to be checked can normally be expanded, It is bad to exclude DNA purity, concentration, or cause PCR to detect the case where failing containing PCR inhibitor etc..

Kit of the invention is provided with internal control gene (Internal simultaneously in EGFR genetic mutation type detection architecture Control, IC) detection architecture.Two kinds of systems are reacted simultaneously in same PCR pipe.Internal control gene be also be different from it is to be checked The house-keeping gene of gene EGFR.It will identify that the probe modification of EGFR genetic mutation template is FAM fluorophor, and will identify internal control The probe modification of gene template is HEX fluorophor.By detecting internal control gene magnification situation (channel HEX), can analyze to be checked Whether DNA can normally be expanded, thus exclude leakage reagent adding or sample, sample contain PCR inhibitor etc. cause PCR detection failure The case where.

When carrying out kit test result judgement, FAM, HEX Air conduct measurement should all be without amplifications (no in negative quality-control product (NC) In typical S type curve.Typical S type curve successively shows as exponential phase, straight line phase and plateau) or without Ct value, positive matter FAM, HEX Air conduct measurement should have amplification (in typical S type curve) and value≤35 Ct in control product (PC)；Otherwise it is assumed that experiment nothing Effect need to repeat to test.

Two, experimental materials and equipment

1. for mutational site design synthesis specific primer and probe

Special primer and probe are designed for EGFR genetic mutation site.By specific primer and probe optimization, so as to Realize highly sensitive and quick detection.

Primer and probe after optimization is as follows:

Forward primer (F): CGTCGCTATC AAGGTGTCG (SEQ ID No:1)

Reverse primer (R): CCTGAGGTTC AGAGCCATG (SEQ ID No:2)

Probe (PB): CCCACACAGC AAAGCAGAAA CTCAC (SEQ ID No:3)

2. detecting the extraction of sample process and DNA

Detection sample can be fresh pathological tissue, paraffin-embedded tissue, whole blood, blood plasma or seroperitoneum.Below only with It is illustrated for paraffin-embedded tissue sample.

Before sample collection, patient without tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKIs) class drug therapy；Since DNA sample quality will affect testing result, the paraffin embedding in DNA sample source should be determined Contain cancerous tissue cell, and the 25% of no less than entire sample in tissue samples；And DNA sample OD260/OD280=1.8 ± 0.2, OD260/OD230 >=1.5；Concentration is 5~10ng/ μ l, is no more than 10ng/ μ l.

Conserving time limit is no more than 3 years paraffin-embedded tissue sample at room temperature, and the DNA sample after extraction is freezed at -20 DEG C Under the conditions of conserving time limit be no more than 6 months.

3. applicable instrument

Stratagene Mx3000P。

4. the composition of kit

Kit forms are shown in Table 1.Nucleic acid extraction composition is free of in kit, uses Autostation N16 nucleic acid purification Analyzer [capital medicine prison tool (standard) 2013 the 1400574th] (Beijing Yakangbo Biotechnology Co., Ltd.'s production) completes paraffin packet Bury tissue samples DNA extraction.According to the operation instruction of nucleic acid purification analyzer, every 8 unions are used to detect 1 sample in kit Product, containing only reference gene detection architecture (channel FAM), B~H holes are contained simultaneously in the position the A hole (respective tube number 1) of detection reagent EGFR genetic mutation detection architecture (channel FAM) and internal control genetic test system (channel HEX).

Table 1: kit forms

Wherein the particular sequence of the reference gene and internal control gene can be by those skilled in the art according to experimental conditions It is readily determined or is provided by Beijing Yakangbo Biotechnology Co., Ltd..

Three, detection methods

1. using Autostation N16 nucleic acid purification analyzer [capital medicine prison tool (standard) 2013 the 1400574th], according to Operation instructions carry out the extraction of paraffin-embedded tissue sample DNA.

2. taking the detection reagent and the positive of corresponding item number (n+2) in kit according to the quantity (n) of DNA sample to be measured Quality-control product PC.Of short duration centrifugation after test agent to be checked and positive quality control product PC melt, is placed on ice.

3. taking the PCR single tube of respective number in kit (n+2) according to the quantity (n) of DNA sample to be measured, Taq is added Archaeal dna polymerase (3 μ l/ pipe) is placed on ice to tube bottom.27 μ l DNA sample to be checked, negative Quality Control are separately added into PCR single tube Product NC (buffer of dissolving DNA) and positive quality control product PC, piping and druming mix, and cover tightly of short duration centrifugation after pipe lid.

Note: not use vortex oscillation instrument when mixing；Subsequent operation is carried out after mixing immediately.

4. the 8 union pipe lids that detection reagent is housed carefully are opened, it is each by being mixed in step 2 with Taq archaeal dna polymerase A DNA sample to be checked, feminine gender quality-control product NC and positive quality control product PC add to the differential responses of detection reagent by 3 holes μ l/ respectively In hole (every 8 unions are for detecting 1 sample).Fluorescent PCR pipe lid is covered tightly, of short duration centrifugation after mixing is flicked.Note: taking, covers The PE or rubber gloves that pipe Gai Shiying more renews；Vortex oscillation instrument is not used when mixing；Reagent is maintained at after of short duration centrifugation Tube bottom and immediately upper machine testing.

5. the setting of fluorescent PCR instrument (Stratagene Mx3000P) sense channel needs simultaneous selection FAM, HEX channel (reference Dyestuff is set as "None").Following (table 2) is arranged in response procedures:

Table 2:

The explanation of four, testing results

1.Ct value determines: set the baseline of Stratagene MX3000P fluorescent PCR instrument first: selection " is suitble to baseline Fluorescence signal when (Adaptive baseline) " is set, threshold value (threshold) setting principle are just above with threshold line The highest point of normal feminine gender quality-control product NC amplification curve (random noise line), even negative quality-control product NC amplification curve is shown Subject to " No Ct ".

Each sample is read from software in the Ct value of each site primer.

2. qualitative analysis:

(1) quality of experiments is judged: if FAM, HEX Air conduct measurement (are not in typical S type curve without amplification in NC.It is typical S type curve successively show as exponential phase, straight line phase and plateau) or without Ct value, FAM, HEX Air conduct measurement have amplification in PC (being in typical S type curve) and value≤35 Ct, then can continue to analyze；Otherwise it is assumed that experiment is invalid, need to repeat to test.

(2) reference gene (IR) detection case is judged in DNA sample to be checked: if reference gene detection (channel FAM) has expansion Increasing and value≤30 22≤Ct, then can continue to analyze；If its Ct value is smaller, then it is assumed that DNA sample excessive concentration；If its Ct value is larger Or without amplification, then it is assumed that DNA sample concentration is too low, degrades, or in which may contain PCR inhibitor.

(3) in DNA sample to be checked internal control genetic test situation judge: if internal control genetic test (channel HEX) have amplification and Value≤35 Ct can then continue to analyze；If internal control genetic test (channel HEX) Ct value is larger or without amplification, but mutational site is detected (channel FAM) has amplification and value≤39 Ct, then can continue analysis (may generate internal control gene magnification since mutational site expands Inhibit)；If internal control genetic test (channel HEX) Ct value is larger or without amplification, and mutational site detection (channel FAM) without amplification or There are amplification but Ct value > 39, then can not continue to analyze, need to repeat experiment (degradation may occur due to DNA sample, wherein contain PCR inhibitor is not loaded product).

(4) gene mutation situation is judged in DNA sample to be checked: if mutational site detection (channel FAM) has amplification in sample And value≤36 Ct, then determine that sample mutation result is the positive；If determining that the sample is mutated result without amplification or Ct value > 39 For feminine gender；If 36 value≤39 < Ct, experiment is repeated, mutation result is doubtful if Ct value still within this range, determines the sample Like positive (Ct value may be caused to fluctuate since mutation content is low).

In an experiment, various mutations be may be simultaneously present in same DNA sample.

Fig. 1 is the PCR figure that testing result is negative sample, and Fig. 2 is the PCR figure that testing result is positive sample.

Pass through contrasting detection, it was demonstrated that fluorescence PCR method of the invention and traditional sequencing methods the result is that be consistent, and this The sensitivity of the fluorescence PCR method of invention and selectivity are higher than traditional sequencing methods.

Therefore, Fluorescence PCR system of the present invention can detect the EGFR gene 2237-2257 mutation for replacing with TGT, inspection Survey convenient and efficient, accuracy is high, can meet the requirement that EGFR genetic mutation quickly detects.

[bibliography]

1.Paez J.G., Janne P.A., Lee J.C. etc., the EGFR mutation in lung cancer: with Gefitinib (gefitinib) association [J] .Science for the clinical response treated, 2004,304 (5676): 1497-1500.

2.Lynch T.J., Bell D.W., Sordella R. etc., in non-small cell lung cancer under Gefitinib response EGF-R ELISA in Activating mutations [J] .N Engl J Med, 2004,350 (21): 2129-2139.

3.Mok T.S., Wu Y.L., Thongprasert S. etc., Gefitinib or carboplatin-taxol in adenocarcinoma of lung [J] .N Engl J Med, 2009,361 (10): 947-957.

4.Kobayashi S., Boggon T.J., Dayaram T. etc., EGFR mutation and non-small cell lung cancer are to lucky non- For tolerance [J] .N Engl J Med of Buddhist nun, 2005,352 (8): 786-792.

5. Li Qi, Zhao Yali, Hao Haojie, Li Xianghong, the mutation research [J] of lung cancer patients in China EGFR gene, China are swollen Tumor magazine, 2007,29:270-273.

6. Song Guohong, Di Lijun, Ren Jun, tension are strong, Yu Jing, Chinese Patients with Non-small-cell Lung EGF-R ELISA The research [J] of gene mutation, modern medical oncology, 2008,16:553-556.

Claims (7)

1. a kind of for detecting the primer of EGFR gene 2237-2257 mutation, which is characterized in that the primer such as SEQ ID Shown in No.1-SEQ ID No.2.

2. a kind of for detecting the probe of EGFR gene 2237-2257 mutation, which is characterized in that the probe such as SEQ ID Shown in No.3.

3. a kind of for detecting the kits of EGFR gene 2237-2257 mutation, which is characterized in that the kit includes Probe shown in primer shown in SEQ ID No.1-SEQ ID No.2 and SEQ ID No.3.

4. kit as claimed in claim 3, which is characterized in that it further includes reference gene and internal control gene.

5. a kind of method of acquisition related Ct value with EGFR gene 2237-2257 mutation comprising following steps:

(1) genomic DNA in sample is extracted；

(2) DNA is expanded with kit as claimed in claim 3；

(3) the FAM fluorescence intensity for detecting reaction system obtains FAM and reaches required cycle-index when the threshold value of setting.

6. method for claim 5, which is characterized in that the sample is fresh pathological tissue, paraffin-embedded tissue, whole blood or blood Starch sample.

7. method for claim 5, which is characterized in that the sample is paraffin-embedded tissue sample.