阅读说明：本技术 一种用于检测ntrk基因融合突变的引物、探针及试剂盒 (A kind of primer, probe and kit being mutated for detecting NTRK Gene Fusion ) 是由 张宇清 赵艳伟 裴婷婷 于 2019-10-30 设计创作，主要内容包括：本发明涉及分子生物技术领域,公开了一种用于检测NTRK基因融合突变的引物、探针及试剂盒。本发明的检测引物、探针分别为SEQ ID NO.1-SEQ ID NO.21所示的序列,探针5’端有FAM或VIC报告荧光基团,3’端有NFQ-MGB淬灭荧光基团。利用本发明可以同时检测6种NTRK基因融合突变,操作简单,灵敏度高,检测速度快,具有良好的临床应用前景。(The present invention relates to field of molecular biotechnology, disclose a kind of for detecting primer, probe and the kit of the mutation of NTRK Gene Fusion.Detection primer of the invention, probe are respectively sequence shown in SEQ ID NO.1-SEQ ID NO.21, and there is FAM or VIC reporter fluorescence group at the end of probe 5 ', and there is NFQ-MGB quenching fluorescence group at 3 ' ends.6 kinds of NTRK Gene Fusions can be detected simultaneously using the present invention to be mutated, easy to operate, high sensitivity, detection speed is fast, has good potential applicability in clinical practice.)

1. a kind of for detecting the primer and probe of the mutation of NTRK Gene Fusion, which is characterized in that primer includes SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.16, shown in SEQ ID NO.17 Nucleotide sequence, probe include SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID Nucleotide sequence shown in NO.15, SEQ ID NO.18, and there is FAM fluorophor at 5 ' ends of probe, 3 ' ends have NFQ-MGB to quench Go out group.

2. primer according to claim 1 and probe, it is characterised in that further include Quality Control primer and Quality Control probe, Quality Control The nucleotide sequence of primer is as shown in SEQ ID NO.19 and SEQ ID NO.20, the nucleotide sequence of Quality Control probe such as SEQ ID Shown in NO.21, there is VIC fluorophor at 5 ' ends of Quality Control probe, and there is NFQ-MGB quenching group at 3 ' ends.

3. primer according to claim 1 or 2 and probe are in preparation for detecting in NTRK Gene Fusion mutagenesis kit Application.

4. application according to claim 3, which is characterized in that the kit can be used in detecting following 6 seed types NTRK Gene Fusion mutation: COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330-TPM3-NTRK1, COSF1447-QKI-NTRK2、COSF1449-NACC2-NTRK2、COSF1332-NTRK1-TPM3。

5. a kind of for detecting the kit of NTRK Gene Fusion mutation, which is characterized in that containing of any of claims 1 or 2 Primer and probe.

6. kit according to claim 5, which is characterized in that containing positive reference substance and negative controls, the sun Property reference substance be claim 4 described in 6 kinds of fusions RNA mixing sample, the negative controls be wanted without right Seek the RNA sample of 6 kinds of fusions described in 4.

7. kit according to claim 5, which is characterized in that further include PCR reaction solution and RNA reverse transcription reaction liquid.

8. kit according to claim 5, which is characterized in that application method the following steps are included:

Extract the RNA in detection sample；

It is cDNA by sample rna, positive reference substance, negative controls the difference reverse transcription of extraction, is carried out using cDNA as template real When fluorescent PCR amplified reaction；

Judged according to the result of fluorescent PCR amplification instrument whether sample occurs fusion mutation.

9. kit according to claim 5, which is characterized in that the kit can be used in detecting following 6 seed types NTRK Gene Fusion mutation: COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330-TPM3- NTRK1、COSF1447-QKI-NTRK2、COSF1449-NACC2-NTRK2、COSF1332-NTRK1-TPM3。

Technical field

The present invention relates to field of molecular biotechnology, more particularly to a kind of for detecting drawing for NTRK Gene Fusion mutation Object, probe and kit.

Background technique

NTRK Gene Fusion mutation belongs to a kind of chromosomal rearrangement, eventually lead to NTRK gene family member (NTRK1, NTRK2, NTRK3) together with other Gene Fusions, result can generate the abnormal T RK fusion protein of conformation activation, and TRK melts Hop protein, which is in, continues active state, causes downstream signal cascade reaction, and the hair of the tumour of TRK Gene Fusion mutation occurs for driving Raw and growth.Research also found, in kinds of tumors, the C-terminal kinase region of NTRK1, NTRK2 or NTRK3 are easy and other bases It is logical so as to cause downstream signal because there are fusion phenomenon, the fusion of these NTRK genes results in the overexpression of fusion protein Road does not depend on the sustained activation of ligand, and then leads oncogenic generation.NTRK Gene Fusion possibly is present at originating from body not With in the tumour of position, including breast cancer, cholangiocarcinoma, colorectal cancer, neuroendocrine carcinoma, non-small cell lung cancer, cancer of pancreas, first Shape gland cancer etc..

On November 26th, 2018, U.S. FDA accelerate approval that sieve is drawn to replace Buddhist nun (Larotrectinib, trade name Vitrakvi) For treating the adult and children's Locally Advanced or the metastatic solid tumors patient that carry NTRK fusion, without considering cancer Happening part.On January 18th, 2019, NCCN have issued non-small cell lung cancer guide 2019 the 3rd edition, non-small cell lung cancer target Become 9 to the related gene of medication from 8, exactly increased NTRK Gene Fusion newly, and draws sieve for Buddhist nun It (Larotrectinib) is even more the recommended Metastatic Nsclc patient positive as the mutation of NTRK Gene Fusion First-line treatment selection.In June, 2019, Roche Holding Ag " unlimited cancer kind " individuation drug grace song replace Buddhist nun (Entrectinib, commodity Name Rozlytrek) Japanese Ministry of Health, Labour and Welfare (MHLW) approval is obtained, for treating NTRK Gene Fusion mutation positive adult's evening Phase recurrent solid tumor patient.These drugs it is granted broken with prodrug must using disease as the boundary of indication, and Opening drug can be using gene mutation as the beginning of indication.Because the use of the two drugs must all be carried with patient Premised on the mutation of NTRK Gene Fusion, therefore quickly and accurately detection NTRK Gene Fusion is mutated as the adjoint of these drugs Diagnosis, it is most important to the selection of clinical of these drugs.

There are immunohistochemical method (IHC), fluorescence in situ hybridization for the detection method of NTRK Gene Fusion mutation at present Technology (FISH), high-flux sequence method and reverse transcriptase polymerase chain reaction (RT-PCR) method etc..IHC method is that applied immunology is anti- The former principle detection tumour specific antigen with antibody specificity ining conjunction with is expressed, and with quick screening effect, but IHC method is directed to It is the expression quantity of albumen, there are certain subjectivities in judgement, and weakly positive result is needed further be verified with technologies such as FISH. FISH technology is the nucleic acid probe homologous complementary using target DNA and fluorescent marker, forms the hybrid of target DNA and nucleic acid probe, It carries out qualitative, quantitative and relative positioning to DNA to be measured under fluorescence microscope to analyze, but the technology is for two genes of fusion When being closer on chromosome location, the separation signal that may cause rearrangement is unobvious, generate false negative result, and cost compared with Height, detection cycle are longer.High-flux sequence method can be used for detection Gene Fusion, but operation is relatively complicated, to experiment The technical requirements of personnel are higher, and time-consuming, usually want 3-5 talent that can go out testing result.Therefore it clinically needs a kind of efficient, high Accuracy rate, high sensitivity, detection method easy to operate detect the mutation of NTRK Gene Fusion, are tumor patient individual Medication provides scientific basis, reduces Operative risk and burden of patients.

In view of the above-mentioned problems, the invention discloses a kind of efficient, high-accuracy, high sensitivity for detecting NTRK gene Merge primer, probe and the kit of mutation.This kit is detection sample with RNA, detects NTRK1, NTRK2, NTRK3 gene With merging for other genes, cover most common 6 kinds of fused types, high sensitivity can be obtained in 150 minutes detection times Testing result, Monitoring lower-cut can achieve 2.5%.

Summary of the invention

The purpose of the present invention is to provide a kind of efficient, high-accuracy, high sensitivity detection NTRK Gene Fusion mutation Primer, probe and kit.

Primer, probe and the kit of a kind of detection NTRK Gene Fusion mutation provided by the invention, which is characterized in that institute State specific primer pair are as follows: nucleotide sequence COSF572-ETV6-NTRK3 as shown in SEQ ID NO.1 and SEQ ID NO.2 Merge amplimer pair, nucleotide sequence COSF1535-ETV6-NTRK3 as shown in SEQ ID NO.4 and SEQ ID NO.5 Merge amplimer pair, nucleotide sequence COSF1330-TPM3-NTRK1 as shown in SEQ ID NO.7 and SEQ ID NO.8 Merge amplimer pair, nucleotide sequence COSF1447-QKI-NTRK2 as shown in SEQ ID NO.10 and SEQ ID NO.11 Merge amplimer pair, nucleotide sequence COSF1449-NACC2- as shown in SEQ ID NO.13 and SEQ ID NO.14 NTRK2 merges amplimer pair, nucleotide sequence COSF1332- as shown in SEQ ID NO.16 and SEQ ID NO.17 NTRK1-TPM3 merges amplimer pair, nucleotide sequence ETV6 Quality Control as shown in SEQ ID NO.19 and SEQ ID NO.20 The amplimer pair of gene.

The specific probe are as follows: nucleotide sequence COSF572-ETV6-NTRK3 as shown in SEQ ID NO.3 fusion Detection probe (5 ' end flag F AM fluorophor), nucleotide sequence COSF1535-ETV6- as shown in SEQ ID NO.6 NTRK3 merges detection probe (5 ' end flag F AM fluorophor), nucleotide sequence COSF1330- as shown in SEQ ID NO.9 TPM3-NTRK1 merges detection probe (5 ' end flag F AM fluorophor), and nucleotide sequence is as shown in SEQ ID NO.12 COSF1447-QKI-NTRK2 merges detection probe (5 ' end flag F AM fluorophor), nucleotide sequence such as SEQ ID NO.15 Shown in COSF1449-NACC2-NTRK2 fusion detection probe (5 ' end flag F AM fluorophor), nucleotide sequence such as SEQ COSF1332-NTRK1-TPM3 shown in ID NO.18 merges detection probe (5 ' end flag F AM fluorophor), nucleotide sequence The detection probe (5 ' end label VIC fluorophor) of the ETV6 Quality Control gene as shown in SEQ ID NO.21.

Further, the specific probe 5 ' is terminal modified a FAM or VIC fluorophor, and 3 ' terminal modified have non-fluorescence quench Go out group NFQ (Non-Fluorescent Quencher), and the group itself does not generate fluorescence, on the one hand can substantially reduce this The intensity of bottom signal, on the other hand, when probe is complete, the fluorescence that reporter group is emitted is quenched group absorptions, instrument Device can't detect signal, and with the progress of PCR, Taq enzyme encounters probe in conjunction with template during DNA chain extends, and 5 ' → 3 ' exonuclease activities will cut off probe, and fluorophor generates fluorescence signal far from quenching group.This is special simultaneously Property probe on be also connected with MGB modification group, can by the Tm value of the probe improve 10 DEG C or so, increase the specificity of probe.

The kit of a kind of detection NTRK Gene Fusion mutation, which is characterized in that comprise the steps of:

1) sample rna extracts

According to sample properties, sample rna extraction is carried out using corresponding RNA extracts kit, will be extracted using nuclease-free water RNA be diluted to that 10-100ng/ μ L is spare, between 1.8~2.0, the RNA after extraction is answered the A260/A280 value of the RNA of extraction It uses immediately, if not having to that temporarily -70 DEG C or less preservations need to be placed on.

2) RNA reverse transcription is cDNA

Reaction solution is respectively configured according to following system in the sample rna of extraction, positive reference substance RNA, negative controls RNA:

Reagent	Volume
		Reverse transcriptase primer (10 μM)	2μL
2.5mM each dNTP Mix	4μL
		5×First-Strand Buffer	4μL
0.1M DTT	1μL
		RNaseOUT Recombinant RNase Inhibitor	1μL
SuperScript III RT	1μL
		Template ribonucleic acid	2μL
Nuclease-free water	Complement to 20 μ L

It is reacted according to following response procedures:

Temperature	Time	Recurring number
			25℃	5min	1
50℃	60min	1
			70℃	15min	1

After reaction, it is placed in immediately at least 2 minutes on ice, obtains cDNA, the cDNA of acquisition is standby as amplified reaction template With.

3) real-time fluorescence PCR detection

In super-clean bench according to following table be respectively configured COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330-TPM3-NTRK1、COSF1447-QKI-NTRK2、COSF1449-NACC2-NTRK2、COSF1332-NTRK1- This six kinds of Gene Fusions of TPM3 detect reaction system:

Reagent	Volume
		2× qPCR mix	10μL
It merges F-Primer (10 μM)	0.4μL
		It merges R-Primer (10 μM)	0.4μL
It merges Probe (10 μM)	0.2μL
		Quality Control gene-F-Primer (10 μM)	0.4μL
Quality Control gene-R-Primer (10 μM)	0.4μL
		Quality Control gene-Probe (10 μM)	0.2μL
50×ROX Dye I	0.4μL
		Template cDNA	2μL
Nuclease-free water	Complement to 20 μ L

It is reacted according to following response procedures:

The fluorescence in the channel FAM, VIC is acquired at 60 DEG C.

Each reaction is respectively provided with positive control and negative control.

The positive reference substance is to contain COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330- This six kinds of TPM3-NTRK1, COSF1447-QKI-NTRK2, COSF1449-NACC2-NTRK2, COSF1332-NTRK1-TPM3 The RNA mixing sample of NTRK Gene Fusion；

The negative controls are without COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330-TPM3- This six kinds of NTRK bases of NTRK1, COSF1447-QKI-NTRK2, COSF1449-NACC2-NTRK2, COSF1332-NTRK1-TPM3 Because of the RNA sample of fusion.

4) interpretation is carried out according to fluorescent PCR amplification

Quality control standard:

A) positive reference substance: there is normal smooth amplification curve in the channel FAM, and there is normal smooth amplification curve in the channel VIC；

B) negative controls: the channel FAM does not have amplification curve, and there is normal smooth amplification curve in the channel VIC.

Meet above-mentioned condition is that Quality Control is qualified.

Pattern detection result interpretation:

A) there is amplification curve in such as channel VIC, and there is amplification curve in the channel FAM, then sample is that the mutation of NTRK Gene Fusion is positive；

B) there is amplification curve in such as channel VIC, and the channel FAM is without amplification curve, then sample is that the mutation of NTRK Gene Fusion is negative or low In this kit Monitoring lower-cut or sample quality reason or be other unlapped fused types of this kit；

C) such as channel VIC does not have amplification curve, then experimental result is insincere, need to re-start experiment.

Therefore, using it is of the present invention it is a kind of detection NTRK Gene Fusion mutation kit, can be used in detection with The NTRK Gene Fusion site of lower 6 seed types: COSF572-ETV6-NTRK3, COSF1535-ETV6-NTRK3, COSF1330- TPM3-NTRK1、COSF1447-QKI-NTRK2、COSF1449-NACC2-NTRK2、COSF1332-NTRK1-TPM3。

The beneficial effects of the present invention are: can specifically detect 6 kinds present invention employs specific primer and probe The mutation of NTRK Gene Fusion.The method: (1) establishing real-time fluorescence PCR system, and realization is mutated NTRK Gene Fusion quick Detection；(2) used time is short, only needs detection can be completed within 150 minutes；(3) testing result interpretation is convenient, high specificity, according to whether there is or not Amplified signal can determine that result；(4) high sensitivity can detect that the Gene Fusion mutation positive plasmid of minimum 25 copies； (5) sample detection range is wide, and sample can be flesh tissue, paraffin-embedded tissue or frozen pathologic section.

Detailed description of the invention

Fig. 1 is that present invention detection positive gene merges reference substance result schematic diagram.

Fig. 2 is that present invention detection negative genes merge reference substance result schematic diagram.

Fig. 3 is the present invention to COSF572-ETV6-NTRK3 Gene Fusion sample sensitivity analysis experimental result schematic diagram.

Fig. 4 is that the present invention illustrates COSF572-ETV6-NTRK3 Gene Fusion pattern detection freight weight limit renaturation experimental result Figure.

Fig. 5 is the experimental result schematic diagram of the present invention 1 positive clinical sample of detection.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Material used in embodiment Material, reagent etc. are unless otherwise specified that can buy the conventional products obtained by market.