CN107630087A - For detecting primer, probe and the kit of 1394 mutation of C-KIT genes - Google Patents

Abstract

The invention discloses a kind of primer, probe and kit for being used to detect 1394 mutation of C KIT genes.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 the kit of the present invention, 1394 C of C KIT genes can be detected and replace with G mutation.

Description

For detecting primer, probe and the kit of 1394 mutation of C-KIT genes

Technical field

The present invention relates to biological technical field, specifically, is related to a kind of for detecting C-KIT Primer, probe and the related kit of gene mutation.

Background technology

C-KIT is a kind of proto-oncogene, positioned at chromosome 4q11-12, total length about 80kb.C-KIT The C-KIT albumen of coding belongs to type III receptor tyrosine kinase family member, it be molecular weight about For 145KD transmembrane protein, EGFR-TK area, transmembrane region and the part knot of intracellular are contained Close site extracellular region.Itself protein tyrosine kinase activity can be activated after part is in connection, Downstream signal transduction path is activated by series reaction, so as to adjust the growth of cell and propagation.

Gastrointestinal stromal tumor (GIST) is most common leaf source property tumour of alimentary canal.Research shows, C-KIT gene mutation rates are about 90% in GIST patient.

Sanger PCR sequencing PCRs are the main methods of C-KIT detection in Gene Mutation at present.However, should The sensitivity of method is low, can cause missing inspection and the generation of false negative, and detection time is grown, nothing Method meets the actual demand of clinical detection.Clinically there is an urgent need to develop a kind of high sensitivity, fast The C-KIT detection in Gene Mutation technologies of speed.

The present inventor is found that in gastrointestinal stromal tumor (GIST) routine testing and stomach Intestinal Stromal Tumors are relevant to dash forward positioned at the new mutation site of C-KIT genes, i.e. 1394 C ＞ G Become.The present inventor develops a kind of quick, highly sensitive, easy to operate for the mutation Detection kit and correlation detection method, can be used for GIST chemotherapy prognosis.

The content of the invention

It is an object of the invention to provide a kind of primer and spy for being used to detect C-KIT gene mutations Pin, the mutation is that 1394 C replace with G (base mutations：1394C ＞ G, protein mutation： S465C).The primer of the present invention includes following sequence with probe：

Forward primer (F)：GATGTGCAGACACTAAACTCACG(SEQ ID No：1)

Reverse primer (R)：GCACTAGAATCTATAGAACTCTGAAC(SEQ ID No：2)

Probe (PB)：TGGGCCACCGTTTGGAAAGCTAG(SEQ ID No：3)

Another aspect of the present invention provides a kind of detection for being used to detect 1394 mutation of C-KIT genes Kit, the kit include SEQ ID No.1-SEQ ID No.2 shown in primer and Probe shown in 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 1394 mutation of detection C-KIT genes, its Comprise the following steps：

(1) genomic DNA in sample is extracted, detection sample includes fresh pathological tissue, paraffin Investing tissue, whole blood and blood plasma etc.；

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

(3) the Ct values shown according to fluorescent PCR instrument judge testing result：Detect reaction system FAM fluorescence intensities, the cycle-index Ct values for reaching required during the threshold value of setting with FAM are made It is feminine gender for negative, Positive judgement standards, Ct values ＞ 38 or without amplification, Ct value≤38 are the positive.

, can be with specific detection C-KIT bases present invention employs specific primer and probe technique Because of mutation.The method high sensitivity, high specificity, detection speed are fast.

Brief description of the drawings

Fig. 1 is the PCR figures of detection negative sample (wild type).

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

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these are implemented Example is only used for the present invention rather than limitation the scope of the present invention.Unreceipted specific bar in embodiment The experimental method of part, according to normal condition well known to those skilled in the art, or according to manufacture Condition recommended by the manufacturer.

One, principles

After the specific primer of the present invention is combined with probe with DNA profiling, Taq archaeal dna polymerases With deoxynucleotide (dNTP) for substrate, to reference gene (Internal Reference, IR) and C-KIT genic mutation types gene carries out amplification in vitro.Detection uses Fluorescence PCR assay, passes through Specific probe hydrolysis release fluorescence, the progress of monitoring PCR reactions, determines that C-KIT genes are dashed forward Change situation.

The kit of the present invention has been separately provided reference gene detection architecture.Reference gene is difference In the house-keeping gene of C-KIT genes to be checked.By the amplification situation (FAM for detecting reference gene Passage), can analyze whether DNA to be checked can normally be expanded, so as to exclude DNA purity, Concentration is bad, or the situation of PCR detection failures is caused containing PCR inhibitor etc..

The kit of the present invention is provided with internal control simultaneously in C-KIT genic mutation type detection architectures Gene (Internal Control, IC) detection architecture.Two kinds of systems in same PCR pipe simultaneously Reacted.Internal control gene is also the house-keeping gene for being different from gene C-KIT to be checked.Will identification The probe modification of C-KIT gene mutation templates is FAM fluorophors, and will identify internal control gene The probe modification of template is HEX fluorophors.By detecting internal control gene magnification situation (HEX Passage), it can analyze whether DNA to be checked can normally be expanded, so as to exclude Lou reagent adding or sample Originally, sample contains the situation that PCR inhibitor etc. causes PCR detection failures.

When carrying out the judgement of this kit testing result, FAM, HEX lead in negative quality-control product (NC) Road detection all should be without amplification (not in typical S types curve.Note：Typical S types curve is successively Show as exponential phase, straight line phase and plateau) or without Ct values, FAM in positive quality control product (PC), HEX Air conduct measurements should have amplification (being in typical S types curve) and Ct value≤28；Otherwise recognize It is invalid to test, it need to repeat to test.

Two, experiment materials and equipment

1. for mutational site design synthesis specific primer and probe

Special primer and probe are designed for C-KIT gene mutation sites.Pass through specific primer And probe optimization, to realize highly sensitive and quick detection.

Primer and probe after optimization is as follows：

Forward primer (F)：GATGTGCAGACACTAAACTCACG(SEQ ID No：1)

Reverse primer (R)：GCACTAGAATCTATAGAACTCTGAAC(SEQ ID No：2)

Probe (PB)：TGGGCCACCGTTTGGAAAGCTAG(SEQ ID No：3)

2. detect sample process and DNA extraction

It can be fresh pathological tissue, paraffin-embedded tissue, whole blood, blood plasma and abdomen to detect sample Chamber hydrops.Only illustrated below by taking paraffin-embedded tissue sample as an example.

Before sample collection, patient is without tyrosine kinase inhibitor (tyrosine kinase Inhibitors, TKIs) class drug therapy；Because DNA sample quality can influence testing result, because This, which should determine that, contains cancerous tissue cell in the paraffin-embedded tissue sample in DNA sample source, and No less than the 25% of whole sample；And DNA sample OD260/OD280=1.8 ± 0.2, OD260/OD230≥1.7；Concentration is 5-10ng/ μ l.

Conserving time limit is no more than 12 months paraffin-embedded tissue sample at room temperature, after extraction DNA sample conserving time limit under -20 DEG C of freezing conditions is no more than 6 months.

3. it is 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 QIAamp (Qiagen companies produce DNA FFPE Tissue Kit, article No.：56404) kit completes stone The sample DNA extraction of wax investing tissue.Detection reagent forms and C-KIT genes mutational site to be checked It is shown in Table 2.IR detection reagents are containing only reference gene detection architecture (FAM passages), C-KIT inspections Test agent is examined containing C-KIT detection in gene mutation system (FAM passages) and internal control gene simultaneously Survey system (HEX passages).

Table 1：Kit forms

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

Three, detection methods

1. QIAamp DNA FFPE Tissue Kit (production of Qiagen companies) kit is used, Paraffin-embedded tissue sample DNA extraction is carried out according to operation instructions.

2. take the detection reagent and positive quality control product (PC) in kit.Test agent and sun to be checked Property quality-control product PC melt after of short duration centrifugation, be placed on ice.

3. volume ratio mixing IR detection reagents and Taq archaeal dna polymerases by 17: 0.3, are pressed 17: 0.3 volume ratio mixing C-KIT detection reagents and Taq archaeal dna polymerases.By 17.3/ Hole dispenses IR detection reagents and C-KIT detection reagents into eight connecting legs.Tried to being detected equipped with IR 2.7 μ l DNA samples to be checked, feminine gender are separately added into eight connecting legs of agent and C-KIT detection reagents Quality-control product (NC, the buffer solution of dissolving DNA, provided for oneself by user) and positive quality control product PC, Piping and druming mixes, and covers tightly of short duration centrifugation after lid.Pay attention to：Vortex oscillation instrument is not used during mixing； Subsequent operation is carried out after mixing immediately.

4. the setting of fluorescent PCR instrument sense channel needs simultaneous selection FAM, HEX passage (reference Dyestuff is arranged to "None").Response procedures are set following (table 2)：

Table 2：

The explanation of four, testing results

1.Ct values determine：The baseline of Stratagene MX3000P fluorescent PCR instrument is set first： Fluorescence signal during selection " being adapted to baseline (Adaptive baseline) " setting, threshold value (threshold) Setting principle is (random to make an uproar just above normal negative quality-control product NC amplification curves with threshold line Sound line) peak, even negative quality-control product NC amplification curves show that " No Ct " are defined. Ct value of each sample in each site primer is read from software.

2. qualitative analysis：

(1) quality of experiments is judged：If FAM, HEX Air conduct measurement (are not in without amplification in NC Typical S type curves.Note：Typical S types curve show as successively exponential phase, the straight line phase and Plateau) or without Ct values, in PC, FAM, HEX Air conduct measurement have amplification (is in typical S Type curve) and Ct value≤28, then it can continue to analyze；Otherwise it is assumed that experiment is invalid, need to repeat real Test.

(2) reference gene (IR) detection case is judged in DNA sample to be checked：If reference gene detects (FAM passages) has amplification and 19≤Ct value≤25, then can continue to analyze；If its Ct value is smaller, Then think DNA sample excessive concentration；If its Ct value is larger or without amplification, then it is assumed that DNA samples Product concentration is too low, degrades, or may wherein contain PCR inhibitor.

(3) internal control genetic test situation is judged in DNA sample to be checked：If internal control genetic test (HEX passages) has amplification and Ct value≤25, then can continue to analyze；If internal control genetic test (HEX Passage) Ct values are larger or without amplification, but mutational site detection (FAM passages) have amplification and Ct value≤38, then can continue analysis (may produce because mutational site expands to internal control gene magnification Suppress)；Internal control genetic test if (HEX passages) Ct values are larger or without amplification, and are mutated position Point detection (FAM passages) can not then continue to analyze without expanding or having amplification but Ct values ＞ 38, Need to repeat experiment (degraded may occur due to DNA sample, wherein containing PCR inhibitor or Product are not loaded).

(4) gene mutation situation is judged in DNA sample to be checked：If certain mutational site is detected in sample (FAM passages) has amplification and Ct value≤38, then judges that sample mutation result is the positive；If Ct values ＞ 38 or without amplification, then judge that sample mutation result is feminine gender.

Pay attention to：Various mutations are may be simultaneously present in same DNA sample.

Fig. 1 is the PCR figures that testing result is negative sample (wild type sample), and Fig. 2 is Testing result is schemed for the PCR of positive sample (saltant type sample).

Pass through contrasting detection, it was demonstrated that fluorescence PCR method of the invention and the knot of traditional sequencing methods Fruit is consistent, and the sensitivity of the fluorescence PCR method of the present invention and selectivity are higher than conventional measurement Sequence method.

Therefore, Fluorescence PCR system of the present invention can detect 1394 C of C-KIT genes and replace G mutation are changed to, easy to detect quick, accuracy is high, can meet that C-KIT gene mutations are quick The requirement of detection.

Claims (5)

1. A kind of 1. primer for being used to detect 1394 mutation of C-KIT genes, it is characterised in that The primer is as shown in SEQ ID No.1-SEQ ID No.2.
2. A kind of 2. probe for being used to detect 1394 mutation of C-KIT genes, it is characterised in that The probe is as shown in SEQ ID No.3.
3. A kind of 3. kit for being used to detect 1394 mutation of C-KIT genes, it is characterised in that The kit includes the primer and SEQ ID No. shown in SEQ ID No.1-SEQ ID No.2 Probe shown in 3.
4. 4. kit as claimed in claim 3, it is characterised in that it also includes reference gene With internal control gene.
5. 5. a kind of method of 1394 mutation of detection C-KIT genes, it comprises the following steps：

   (1) genomic DNA in sample is extracted；

   (2) DNA is expanded with the kit described in claim 3；

   (3) the Ct values shown according to fluorescent PCR instrument judge testing result.