CN112609001A - Primer, probe and method for detecting TP63-NTRK2 fusion gene and relative expression level thereof - Google Patents

Abstract

The invention discloses a primer, a probe and a method for detecting a TP63-NTRK2 fusion gene and the expression level of the fusion gene relative to an NTRK2 gene in a tumor patient by utilizing a fluorescent quantitative PCR technology, which can screen whether the TP63-NTRK2 fusion gene exists in the tumor patient, and detect the relative expression quantity of the NTRK2 gene, and have important significance for timely adjusting a treatment scheme, guiding the use of erlotinib, evaluating the treatment effect, predicting prognosis and preventing clinical relapse of the tumor patient.

Description

Primer, probe and method for detecting TP63-NTRK2 fusion gene and relative expression level thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to a method for screening fusion genes, which adopts a probe real-time fluorescence PCR technology to detect TP63-NTRK2 fusion genes and expression levels of the fusion genes relative to NTRK2 genes in tumor patients.

Background

Malignant tumor is the first cause of death of diseases in the world at present, and seriously threatens the health of human beings. The NTRK fusion is an oncogenic driver gene, the TP63-NTRK2 fusion gene is formed by fusion between the 6 th exon of the TP63 gene and the 12 th exon of the NTRK2 gene, the fusion product is chimeric protein, and plays a role in non-ligand-dependent constitutive activation to trigger a permanent signal cascade reaction and drive the growth of cancer cells. The NTRK gene fusion type is found in a variety of solid tumors in adults and children. It has been reported that the frequency of NTRK gene fusion is much lower than 5% in more common malignancies such as lung cancer and colorectal cancer, while the frequency of gene fusion is higher in rare tumor types such as congenital infantile fibrosarcoma, congenital mesodermal renal carcinoma, secretory breast cancer, salivary gland mammary gland-like secretory carcinoma. Other solid tumors also include brain malignancies, pancreatic cancer, melanoma, thyroid cancer, gastrointestinal stromal tumors, etc., with a frequency of NTRK2 gene fusion of less than 1% in all solid tumors.

In recent years, with the development of technology, the individual precise medical treatment taking tumor driving genes as targets thoroughly changes the treatment pattern of malignant tumors, and more molecular targeted drugs are emerging continuously. Larotinib (larotretinib) is a novel oral small molecule, highly selective inhibitor of Tropomyosin Receptor Kinase (TRK). The larotinib is mainly competitively combined with an ATP site of TRKB (coded by NTRK 2) in cells, so that the catalytic activity and autophosphorylation of TRK are inhibited, and downstream signal path conduction is blocked, thereby playing the role of resisting tumors.

Therefore, the method for detecting the relative expression quantity of the TP63-NTRK2 fusion gene and the NTRK2 gene in the tumor patient has important significance for timely adjusting a treatment scheme, guiding the administration of the erlotinib, evaluating the treatment effect, predicting the prognosis and preventing clinical relapse of the tumor patient.

Currently, the methods for detecting NTRK gene fusion are mainly Immunohistochemistry (IHC), Fluorescence In Situ Hybridization (FISH), second generation sequencing (NGS), and reverse transcription polymerase chain reaction (RT-PCR). Although the immunohistochemical method (IHC) is simple in principle, the test process is too complicated, the required reagents are various, the test result needs to be interpreted by experts with rich experience, the interpreted result has high subjectivity, and the judgment of the weak positive result needs to be further verified by technologies such as FISH; fluorescence In Situ Hybridization (FISH) is considered to be a gold standard for detecting gene fusions. The kit has the advantages of high stability, high sensitivity, good specificity, short experimental period and the like, but can only detect one target at a time, needs special tissue and multi-target part detection, does not have a commercialized probe at present, has obvious False Positive (FP) or False Negative (FN), can only detect from a DNA layer, and has high detection cost; the high-throughput sequencing technology can be used for sequencing millions of short sequences in parallel, and is widely applied to detection of tumor mutation, however, the current NGS method only judges the DNA sequencing, and is easy to cause missed detection on the RNA level; compared with the complex data processing in the later stage of the second-generation sequencing, the real-time fluorescent quantitative PCR detection result is visual and easy to judge, and the method has the characteristics of simple operation, low cost and the like. The research adopts a real-time fluorescence quantitative PCR technology, has the advantages of good specificity, high sensitivity, good linear relation, simple operation, high automation degree, pollution prevention, larger linear range and the like, can detect tiny residual focuses, provides basis for solving the progress of the disease and the treatment effect, and simultaneously has the defects of high false positive and false negative rate, influence on detection due to poor primer design and the like in the real-time fluorescence quantitative PCR. However, the technology is mature in detection, practical, reliable and stable in result, and can be used as a first-choice method for detecting the relative expression quantity of TP63-NTRK2 gene fusion and NTRK2 gene, and used for guiding the administration of the erlotinib, evaluating the treatment effect and predicting prognosis. Common methods in real-time fluorescent quantitative PCR include SYBR GreenI dye method, double-probe hybridization method, Taqman technology and the like. Wherein, SYBR GreenI is unsaturated dye, so the specificity is not as good as that of a double-probe hybridization method and a Taqman method, and the specificity is judged by observing a dissolution curve; the two-probe hybridization method is expensive. Therefore, the research adopts a real-time fluorescent PCR technology combined with a Taqman probe method to be applied to the TP63-NTRK2 gene fusion and the detection of the relative expression quantity of the NTRK2 gene.

Previous studies on fusion genes focused only on the conditions of the fusion gene itself, but did not consider the expression conditions of the relevant normal genes, and the level of the expression level of the fusion gene relative to the expression level of the normal gene could not be known.

Disclosure of Invention

The invention designs a primer and a probe sequence for detecting an internal reference/target gene, and screens a TP63-NTRK2 fusion gene and the expression level of the fusion gene relative to an NTRK2 gene in a patient body by adopting a real-time fluorescent PCR technology. The method is rapid and accurate, and has high sensitivity, good specificity and large detection flux. The invention provides the expression level of the normal gene as reference, and can provide more references for the clinical medication and related treatment of the erlotinib.

The test reagent for screening the TP63-NTRK2 fusion gene and the expression level of the fusion gene relative to the NTRK2 gene in a patient comprises erythrocyte lysate, TRIzol, chloroform, absolute ethyl alcohol, ReverTra AceqPCR RT Kit (TOYOBO company), a detection system PCR reaction solution, a positive control product and a negative control product.

The PCR reaction solution of the detection system comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101) and upstream and downstream primers for detecting target genes, wherein the upstream and downstream primers are respectively as follows: TP63-E6-F, NTRK2-E11-F, NTRK2-E12-R, a probe is NTRK2-E12-P, a primer for detecting the reference gene Actin is Actin-F and Actin-R, and the probe is Actin-P. Wherein,

TP63-E6-F：GACAGGAAGGCGGATGAAGA

NTRK2-E12-R：AGTGGGCTGGCAGAGTCATC

NTRK2-E12-P：FAM-TCATTGCTGATAACGGAGGCTGGG-TAMRA

NTRK2-E11-F：TTTTGGTAATGCTGTTTCTGCTT

Actin-F:TGAGCGAGGCTACAGCTT

Actin-R:TCCTTGATGTCGCGCACGATTT

Actin-P:FAM-ACCACCACGGCCGAGCGG-TAMRA。。

specifically, the positive control substances are solutions containing TP63-NTRK2 fusion gene and NTRK2 gene respectively; the negative control product does not contain TP63-NTRK2 fusion gene and NTRK2 genome solution.

The invention also provides a method for detecting the TP63-NTRK2 fusion gene and the expression level of the fusion gene relative to the NTRK2 gene, which comprises the following steps:

1) extracting total RNA in tissues and carrying out reverse transcription to obtain cDNA;

2) detecting whether a TP63-NTRK2 fusion gene exists in a sample by using the cDNA in the step 1 as a template and an upstream/downstream primer TP63-E6-F/NTRK2-E12-R and a probe NTRK2-E12-P for detection; detecting the relative expression quantity of NTRK2 genes in a sample by using an upstream/downstream primer NTRK2-E11-F/NTRK2-E12-R and a probe NTRK2-E12-P for detection; quantitatively detecting the expression quantity of the Actin in a sample by using an internal reference gene upstream/downstream primer Actin-F/Actin-R Probe Actin-Probe;

3) analyzing whether the sample has TP63-NTRK2 gene fusion and the expression level of the relative NTRK2 gene according to the Real-time PCR result;

in the step 1), the extraction method is that blood RNA is extracted by a conventional TRIZOL method, and then the blood RNA is reversely transcribed into cDNA by a Rever Tra Ace qPCR RT Kit of TOYOBO company.

The Real-time PCR amplification method in the step 2) is as follows:

one sample qPCR system was 25 ul:

2 x qPCR MIX 12.5ul, ROX Reference Dye (50 x) 0.5ul, primers TP63-E6-F, NTRK2-E12-R each 0.4ul, probe NTRK 2-E12-P0.2 ul, sterile water 7.8ul, cDNA template 2 ul.

2 x qPCR MIX 12.5ul, ROX Reference Dye (50 x) 0.5ul, primers NTRK2-E11-F, NTRK2-E12-R each 0.4ul, probes NTRK 2-E12-P0.2 ul, sterile water 7.8ul, cDNA template 2 ul.

The qPCR system for the internal controls was 25 ul:

2 x qPCR MIX 12.5ul, ROX Reference Dye (50 x) 0.5u, each of primers Actin-F, Actin-R0.4 ul, Probe Actin-Probe 0.4ul, sterile water 8.8ul, cDNA template 2 ul.

Real-time PCR reaction program: pre-denaturation at 95 ℃ for 1 min; the temperature is 95 ℃ for 15s, and the temperature is 58 ℃ for 35s, and the circulation is carried out for 40 times.

Analyzing the result of the step 3), specifically comprising the following steps: the Real-time PCR of internal reference (Actin) and target genes TP63-NTRK2 and NTRK2 was performed simultaneously on one plate:

a. when the internal reference is positive, the detection result is considered to be effective;

b. positive judgment standard: ct <36, positive, TP63-NTRK2 gene fusion in the sample; ct is more than or equal to 36 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative, no TP63-NTRK2 gene fusion in the sample.

The invention also provides a kit for detecting the expression level of the TP63-NTRK2 fusion gene and the relative NTRK2 gene, which comprises a detection system PCR reaction liquid, wherein the PCR reaction liquid comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101), and upstream and downstream primers for detecting the target gene are respectively as follows: TP63-E6-F, NTRK2-E11-F, NTRK2-E12-R, a probe is NTRK2-E12-P, a primer for detecting the reference gene Actin is Actin-F and Actin-R, and the probe is Actin-P.

The invention has the beneficial effects that: firstly, the invention not only screens TP63-NTRK2 fusion gene in a patient body, but also detects the expression level of the fusion gene relative to NTRK2 gene, thereby providing more references for the clinical medication and related treatment of the erlotinib. The TP63-NTRK2 fusion gene and the NTRK2 gene share the same downstream primer and probe, so that the experiment cost is saved. And thirdly, the primers and the probes required by the reaction system are reasonably proportioned and optimized, so that the experimental conditions are optimal, a complicated condition groping link is omitted, and the experimental efficiency is greatly improved. The method has good specificity, high sensitivity and simple operation. The kit is beneficial to monitoring the expression level of the TP63-NTRK2 fusion gene and the relative NTRK2 gene in a clinical tumor patient, and has important significance for guiding the administration of the erlotinib, evaluating the treatment effect and predicting the prognosis.

Drawings

FIG. 1 is a graph showing the amplification curves of 20 samples, TP63-NTRK2 positive samples and negative controls, using the primer probes and methods of the present invention.

FIG. 2 is a graph showing the amplification curves of 20 samples, NTRK2 positive samples and negative controls tested using the primer probes and methods of the invention.

Detailed Description

The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.

Example 1

The invention is used for assisting the diagnosis of the TP63-NTRK2 fusion gene in a tumor patient body clinically and establishing a personalized treatment scheme. The reagent comprises: erythrocyte lysate, TRIzol, chloroform, absolute ethanol, ReverTra Ace qPCR RT Kit (TOYOBO Co.).

Detection system PCR reaction solution: ReverTra Aceq PCR RT Kit (TOYOBO Co.); the primer and Probe concentrations of the THERNDERBIRD Probe qPCR Mix (2X), the Actin reference gene and the TP63-NTRK2 target gene are all 10 mu M; wherein, the primers and probes for detecting the reference gene ABL and the target gene RET-PTC are respectively as follows:

TP63-E6-F：GACAGGAAGGCGGATGAAGA；

NTRK2-E12-R：AGTGGGCTGGCAGAGTCATC；

NTRK2-E12-P：FAM-TCATTGCTGATAACGGAGGCTGGG-TAMRA；

NTRK2-E11-F：TTTTGGTAATGCTGTTTCTGCTT；

Actin-F:TGAGCGAGGCTACAGCTT；

Actin-R:TCCTTGATGTCGCGCACGATTT；

Actin-P:FAM-ACCACCACGGCCGAGCGG-TAMRA；

positive control: solutions containing TP63-NTRK2 fusion gene and NTRK2 gene respectively; negative control: the solution without TP63-NTRK2 fusion gene and NTRK2 gene.

Example 2

The method comprises the following operation processes:

(1) the tissue was ground in liquid nitrogen. Adding 1ml of lysis solution RZ into 50-100mg of tissue, and homogenizing with a homogenizer. The sample volume should not exceed one tenth of the RZ volume of the lysate. The homogenate sample was left at 15-30 ℃ for 5min to allow complete separation of the nucleic acid-protein complex. Centrifugation was carried out at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 5min, and the supernatant was removed and transferred to a new RNase-free centrifuge tube. Add 200. mu.l chloroform, cover the tube, shake vigorously for 15sec, and let stand at room temperature for 3 min. Upon centrifugation at 12,000rpm (13,400 Xg) for 10min at 4 ℃, the sample will separate into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the used lysis solution RZ reagent. The aqueous phase was transferred to a new tube. 0.5 volume of absolute ethanol was added slowly and mixed well (precipitation may occur). The resulting solution was transferred together with the precipitate to an adsorption column CR3 and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, and if the whole solution and mixture could not be added to the adsorption column CR3 at one time, it was transferred to an adsorption column CR3 in two portions and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, and the waste liquid in the collection tube was discarded. To the adsorption column CR3, 500. mu.l of deproteinized solution RD (previously checked for the presence of ethanol) was added, centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, the waste liquid was discarded, and CR3 was placed in a collection tube. To the adsorption column CR3 was added 500. mu.l of the rinsing solution RW (please first check whether ethanol was added or not), the mixture was allowed to stand at room temperature for 2min, centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, and the waste solution was discarded. Repeating the previous operation steps. The adsorption column was placed in a 2ml collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 2min to remove residual liquid. The adsorption column CR3 was transferred to a new 1.5ml centrifuge tube, 30-100. mu.l RNase-Free ddH2O was added, the mixture was left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 2 min.

(2) RNA was inverted to cDNA with reference to the ReverTra Ace qPCR RT Kit instructions from TOYOBO.

(3) Reagent preparation: preparing X ul of PCR reaction liquid of a detection system according to the number of detected persons, and subpackaging 23ul of each person:

x ═ 23ul reaction X (n specimens +1 positive control +1 negative control +1 blank);

(4) sample adding: adding 2ul of cDNA into the PCR reaction solution of the detection system; 2ul of positive control and negative control are directly added into the positive control and the negative control; blank control was added with 2ul of physiological saline or no substance.

(5) And (3) detection: the detection was performed on a real-time fluorescent PCR instrument, and available instruments include ABI7300, 7500 (Applied Biosystems, USA), and the like. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 58 ℃ for 35sec for 40 cycles, and fluorescence signals were collected at 58 ℃ for 35 sec.

(6) And (5) judging a result: the threshold line is adjusted to be above the background signal and the negative amplification line, and the system judges according to the CT value.

1) When the internal reference is positive, the detection result is considered to be effective;

2) positive judgment standard: ct <36, positive; ct is more than or equal to 36 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.

Example 3

Detection of clinical specimens Using the nucleic acid detection method of the invention

20 clinical tumor samples to be detected are taken, and genome is extracted, a reagent is prepared and detected according to the method described in the embodiment 2. Each sample was added to 2. mu.L of the detection system PCR reaction solution. And simultaneously, making positive, negative and blank controls. Each sample was replicated 2 times, 3 positive controls, 1 negative control, and the detection time was only 60 minutes.

All samples in the 20 screened samples were lined with Actin, indicating that the samples were normally available. All samples have NTRK2 gene expression, but the expression level is low, and the reference data shows that the NTRK2 gene has expression in normal tissues of the brain and the thyroid gland and has extremely low expression level in other tissues, which is consistent with the experimental result. No line was drawn in 20 test samples except 3 positive control lines of the TP63-NTRK2 fusion gene. The results of the experiment are shown in table 1, fig. 1 and fig. 2:

TABLE 120 clinical samples TP63-NTRK2 expression levels

Sequence listing

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Claims (7)

1. The primer and the probe for detecting the expression level of the TP63-NTRK2 fusion gene and the relative NTRK2 gene in a tumor patient are characterized in that the primer and the probe for amplifying the TP63-NTRK2 fusion gene and the NTRK2 gene are respectively as follows:

TP63-E6-F：GACAGGAAGGCGGATGAAGA

NTRK2-E12-R：AGTGGGCTGGCAGAGTCATC

NTRK2-E12-P：FAM-TCATTGCTGATAACGGAGGCTGGG-TAMRA

NTRK2-E11-F：TTTTGGTAATGCTGTTTCTGCTT。

2. the primers and probes for detecting the expression level of the TP63-NTRK2 fusion gene and the relative NTRK2 gene in a tumor patient according to claim 1, further comprising primers and probes for amplifying an Actin reference gene, wherein the primers and probes are respectively as follows:

Actin-F：TGAGCGAGGCTACAGCTT

Actin-R：TCCTTGATGTCGCGCACGATTT

Actin-P：FAM-ACCACCACGGCCGAGCGG-TAMRA。

3. the primers and probes as claimed in claim 1, wherein the ratio of TP 63-E6-F: NTRK 2-E12-R: the molar ratio of NTRK2-E12-P is 2: 2: 1.

4. the primers and probes as claimed in claim 1, wherein NTRK 2-E11-F: NTRK 2-E12-R: the molar ratio of NTRK2-E12-P is 2: 2: 1.

5. the primers and probes according to claim 1, characterized in that the activity of Actin-F: Actin-R: the molar ratio of the Actin to the P is 1: 1: 1.

6. a method for detecting the expression level of TP63-NTRK2 fusion gene and relative NTRK2 gene in tumor patients, comprising the following steps:

(1) extracting total RNA in tissues and carrying out reverse transcription to obtain cDNA;

(2) detecting whether a TP63-NTRK2 fusion gene exists in a sample by using the cDNA in the step 1 as a template and an upstream/downstream primer TP63-E6-F/NTRK2-E12-R and a probe NTRK2-E12-P for detection; detecting the relative expression quantity of NTRK2 genes in a sample by using an upstream/downstream primer NTRK2-E11-F/NTRK2-E12-R and a probe NTRK2-E12-P for detection; quantitatively detecting the expression level of the Actin in a sample by using an internal reference gene upstream/downstream primer Actin-F/Actin-R Probe Actin-Probe, wherein,

TP63-E6-F：GACAGGAAGGCGGATGAAGA

NTRK2-E12-R：AGTGGGCTGGCAGAGTCATC

NTRK2-E12-P：FAM-TCATTGCTGATAACGGAGGCTGGG-TAMRA

NTRK2-E11-F：TTTTGGTAATGCTGTTTCTGCTT

Actin-F：TGAGCGAGGCTACAGCTT

Actin-R：TCCTTGATGTCGCGCACGATTT

Actin-P：FAM-ACCACCACGGCCGAGCGG-TAMRA；

(3) and analyzing whether the sample has TP63-NTRK2 gene fusion and the expression level of the TP63-NTRK2 gene relative to the NTRK2 gene according to the Real-time PCR result.

7. The method according to claim 6, wherein the analysis of the results of step (3) is specifically: the Real-time PCR of internal reference (Actin) and target genes TP63-NTRK2 and NTRK2 was performed simultaneously on one plate:

1) when the internal reference is positive, the detection result is considered to be effective;

2) positive judgment standard: ct <36, positive, TP63-NTRK2 gene fusion in the sample; ct is more than or equal to 36 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative, no TP63-NTRK2 gene fusion in the sample.

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