CN117230204A - Primer probe set, kit and detection system for detecting MET gene 14 exon splicing mutation - Google Patents

Abstract

The invention relates to the technical field of gene detection, in particular to a primer probe set, a kit and a detection system for detecting MET gene 14 exon splicing mutation, wherein the system comprises an acquisition module for acquiring a plasma sample; an extraction module for extracting a free RNA sample from a plasma sample; the amplification module is used for carrying out PCR amplification on the free RNA sample by adopting a primer probe group or a kit; the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample; the analysis module is used for analyzing according to the fluorescence detection result; if the internal reference gene is amplified and the Ct value is less than or equal to 35, the MET14 locus is amplified and the Ct value is less than or equal to 39, the detection result is that the sample MET14 gene jump mutation is positive; the invention realizes the rapid detection of MET14 gene mutation, and has low cost, high efficiency and low analysis complexity.

Description

Primer probe set, kit and detection system for detecting MET gene 14 exon splicing mutation

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer probe set, a kit and a detection system for detecting MET gene 14 exon splicing mutation.

Background

The jump mutation of the MET exon 14 is an independent driving factor of non-small cell lung cancer, when the mutation occurs, the MET gene is transcribed to obtain mRNA with the exon 14 deleted, and truncated MET receptor is generated after translation, and is not easy to be degraded by ubiquitination, so that the duration of the MET is prolonged, the continuous activation of downstream signals is caused, and the occurrence and development of lung cancer are driven.

Because MET jumps occur in DNA introns, which cover a large number of repeated or high GC sequences, sequencing is poor. Detection of MET14 jumps based on RNA levels may be more comprehensive than DNA levels, but RNA detection requires stringent tissue preservation and extraction conditions, and susceptibility to degradation is also a pain point limiting RNA detection. RNA-based detection can increase the detection rate of MET14 exon-skipping mutations.

The real-time quantitative reverse transcription PCR (RT-qPCR) method is a detection technology combining Reverse Transcription (RT) of RNA and real-time quantitative polymerase chain amplification (qPCR) of cDNA, and can detect the mRNA content of the MET exon 14 jump mutation.

The detection principle is that firstly, total RNA in a patient sample is extracted, mRNA in the sample is taken as a template, a primer capable of aiming at MET gene mRNA is added, target mRNA is transcribed into cDNA under the action of reverse transcriptase, then RT-qPCR amplification is carried out by taking the cDNA as the template, a large amount of MET cDNA is obtained, and finally, whether the MET exon 14 is mutated or not is detected, so that the mutation condition of MET is judged. The accuracy and detection rate of mRNA will be higher than that of DNA, which may have hundreds of base mutations, and commercial primers may not cover all mutant forms, thus possibly leading to false negative detection results. Whereas mRNA has only a form in which exons 13 and 15 are fused, it must be detectable if no degradation occurs.

Because of the wide space-time heterogeneity of tumors, sampling of tumor tissue may result in tissue detection results that do not reflect the general appearance of the tumor, and in advanced cancer patients with unavailable partial tissue samples, the opportunity for targeted therapy may also be missed due to the inability to perform tissue biopsies. In addition, tissue sample processing is under-normative, resulting in an unacceptable targeted therapy to and benefit from this portion of the patient. The circulating tumor RNA (ctRNA) can reflect in-vivo tumor load in a short time, dynamically monitors the drug curative effect in real time, can be used as an effective supplement for tissue sample detection, is beneficial to avoiding the detection of false negative results of tissue samples caused by tumor heterogeneity, is easy to obtain due to liquid biopsy samples, can dynamically monitor and manage the curative effect of a late cancer patient incapable of obtaining the tissue samples through ctRNA, optimizes a treatment scheme, and has important significance in relieving clinical symptoms of the late cancer patient, prolonging the life span and improving the life quality.

Methods for detecting the jump mutation of MET14 include second generation sequencing or qRT-PCR to directly detect mRNA lacking MET14 exon, or second generation sequencing to detect genetic variations at the DNA level that may result in cleavage of MET14 exon. The second generation sequencing method can simultaneously detect a plurality of samples, but has the advantages of higher cost, longer experimental period and complex data analysis.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to solve the problems that: how to provide a primer probe set, a kit and a detection system for detecting MET gene 14 exon splicing mutation, so as to solve the problems of high cost, long experimental period and complex data analysis existing in the existing method for detecting MET14 exon-skipping mutation.

In order to solve the problems, the invention adopts the following technical scheme:

in a first aspect, the invention provides a primer probe set for detecting MET gene 14 exon splice mutation in peripheral blood circulation tumor RNA, which comprises primers and probes with nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 3.

As one embodiment, the kit further comprises primers and probes with nucleotide sequences shown as SEQ ID NO.4-SEQ ID NO. 6.

In a second aspect, the invention provides a kit for detecting the splicing mutation of the exon 14 of the MET gene in peripheral blood circulation tumor RNA, which comprises the primer probe group for detecting the splicing mutation of the exon 14 of the MET gene in peripheral blood circulation tumor RNA.

As one embodiment, it comprises 2 parts by volume Buffer SG 60, 4 parts by volume 1M trehalose, 0.6 parts by volume DMSO, 0.2 parts by volume 10 mg/mL BSA, 0.16 parts by volume dNTP, 0.1 parts by volume dUTP, 0.16 parts by volume ROX, 0.34 parts by volume 0.1M MgCl2, 0.5 parts by volume enhancement, 0.4 parts by volume Taq, 0.8 parts by volume reverse transcriptase, 0.5 parts by volume Heat-enzyme nucleotide-DNA glycylase, 0.24 parts by volume non-ribozyme water, 1 part by volume primer of nucleotide sequence shown in SEQ ID No.1, 1 part by volume primer of nucleotide sequence shown in SEQ ID No.2 and 0.5 parts by volume probe of nucleotide sequence shown in SEQ ID No. 3;

or, 2 parts by volume of Buffer SG 6, 4 parts by volume of 1M trehalose, 0.6 parts by volume of DMSO, 0.2 parts by volume of 10 mg/mL BSA, 0.16 parts by volume of dNTPs, 0.1 parts by volume of dUTP, 0.16 parts by volume of ROX, 0.34 parts by volume of 0.1M MgCl2, 0.5 parts by volume of an Enhancer, 0.4 parts by volume of Taq, 0.8 parts by volume of reverse transcriptase, 0.5 parts by volume of Heat-enzyme Uracil-DNA Glycosylase, 0.24 parts by volume of nuclease-free water, 1 part by volume of a primer of a nucleotide sequence shown as SEQ ID NO.4, 1 part by volume of a primer of a nucleotide sequence shown as SEQ ID NO.5 and 0.5 parts by volume of a probe of a nucleotide sequence shown as SEQ ID NO.6 are also included.

In a third aspect, the invention provides an application of the primer probe set for detecting the MET gene 14 exon splice mutation in peripheral blood circulation tumor RNA or the kit for detecting the MET gene 14 exon splice mutation in peripheral blood circulation tumor RNA in preparation of a MET gene 14 exon splice mutation detection reagent.

In a fourth aspect, the invention provides a detection system for detecting a splicing mutation of a MET gene 14 exon in peripheral blood circulation tumor RNA, comprising an acquisition module, an extraction module, an amplification module, a fluorescence detection module and a judgment module;

the collection module is used for collecting a plasma sample;

the extraction module is used for extracting a free RNA sample from the plasma sample;

the amplification module is used for carrying out PCR amplification on the free RNA sample by adopting the primer probe group or the kit;

the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;

and the analysis module is used for analyzing according to the fluorescence detection result.

As an embodiment, the reaction conditions for the PCR amplification in the amplification module are: reverse transcription is carried out for 20min at 55 ℃; activating at 95 ℃ for 5min; cycling for 45 times at 95 ℃, 15s,65 ℃, 5s,56 ℃ and 35 s; kept at 25℃for 10s.

In the analysis module, if the internal reference gene is amplified and the Ct value is less than or equal to 35, the MET14 locus is amplified and the Ct value is less than or equal to 39, the detection result is that the splicing mutation of the MET14 gene of the sample is positive; if the internal reference gene is amplified and the Ct value is less than or equal to 35, and the MET14 locus is not amplified or the Ct value is more than 39, the detection result is that the splicing mutation of the MET14 gene of the sample is negative.

In a fifth aspect, the invention provides a method for detecting a non-disease diagnostic purpose of detecting a MET gene 14 exon splice mutation in peripheral blood circulating tumor RNA, comprising:

collecting a plasma sample;

extracting a free RNA sample from the plasma sample;

carrying out PCR amplification on the free RNA sample by adopting the primer probe group or the kit;

performing fluorescence detection on the amplified sample;

analyzing according to the fluorescence detection result;

if the internal reference gene is amplified and the Ct value is less than or equal to 35, the MET14 locus is amplified and the Ct value is less than or equal to 39, the detection result is that the splicing mutation of the MET14 gene of the sample is positive; if the internal reference gene is amplified and the Ct value is less than or equal to 35, and the MET14 locus is not amplified or the Ct value is more than 39, the detection result is that the splicing mutation of the MET14 gene of the sample is negative.

The invention has the beneficial effects that: the primer probe set, the kit and the detection system for detecting the splicing mutation of the MET gene 14 exon are provided by the invention, an upstream primer, a downstream primer and a specific probe are designed aiming at the MET gene 14 exon, the primer can specifically amplify a section of sequence of the MET gene, the probe can be specifically combined with the MET, the primer and the probe of an internal reference gene are designed, RT-qPCR amplification is adopted, reverse transcription and qPCR reaction are completed in one tube, no additional tube opening/pipetting operation is needed, the RT-PCR detection speed is higher, the detection result can be obtained in 2 working days, and the detection sensitivity is better.

Drawings

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings, in which:

FIG. 1 is a graph showing amplification of a positive plasmid standard according to an embodiment of the present invention;

FIG. 2 is a graph showing amplification of a negative plasmid standard according to an embodiment of the present invention;

FIG. 3 is a graph showing positive amplification of MET14 gene in a sample according to an embodiment of the present invention;

FIG. 4 is a graph showing the negative amplification of MET14 gene in a sample according to the embodiment of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It should be noted that these examples are only for illustrating the present invention, and not for limiting the present invention, and simple modifications of the method under the premise of the inventive concept are all within the scope of the claimed invention.

The design idea of the invention is as follows:

the method designs a specific primer and a probe for detecting the MET14 cDNA, and combines a specific internal reference gene, the primer, the probe and PCR conditions thereof to detect the jump mutation of the MET exon 14. The method aims at establishing a rapid and accurate detection method for the MET exon 14 jump mutation in human peripheral blood circulation tumor RNA, and lays a foundation for improving the biological clinical detection level and popularizing and applying.

The technical scheme adopted by the invention is as follows:

the method comprises the steps of designing an upstream primer, a downstream primer and a specific probe aiming at MET gene RNA, wherein the primer can specifically amplify a sequence of the MET gene, and the probe can specifically bind with MET.

The invention designs an internal reference, and designs an upstream primer, a downstream primer and a specific probe aiming at internal reference gene ACTB gene RNA, wherein the primer can specifically amplify a section of sequence of the ACTB gene, and the probe can specifically combine with the ACTB.

The specific primers and probes are shown in Table 1.

Table 1 primer probe and nucleotide sequence table

RT-qPCR

Reverse transcription and qPCR reactions are completed in one tube, without additional open tube/pipetting operations, greatly improving the detection throughput and reducing the risk of contamination. dUTP/UDG anti-theft agent is introduced into the kit

The system is contaminated. Heat-sensitive (UDG) can degrade U-containing pollutants rapidly at room temperature; when reverse transcription is carried out at 55 ℃, the Heat-enzyme UDG is rapidly inactivated, and the efficiency and the sensitivity of RT-qPCR are not affected. The superior performance of reverse transcriptase and Taq DNA Polymerase of hot start is integrated, and the detection sensitivity of the kit can reach 0.1 pg total RNA or 10 copies of RNA template by matching with an optimized buffer system. The method is suitable for a high-specificity detection system of fluorescent marked probes such as TaqMan and the like.

Preparation of negative and positive standard substance

And (3) amplifying gene fragment sequences by PCR according to the kit, artificially synthesizing MET14 and ACTB target RNA fragments, diluting the cDNA at different concentrations, and finally obtaining a positive reference, wherein the target fragment clone PUC57 is synthesized by a carrier and purchased from the division of biological engineering (Shanghai).

And (3) amplifying a gene fragment sequence by PCR according to the kit, artificially synthesizing an ACTB target RNA fragment, diluting the cDNA at different concentrations, and finally obtaining a negative reference product, wherein the target fragment clone PUC57 is synthesized by a carrier and purchased from a biological engineering (Shanghai) stock company.

Example 1

The process for detecting MET gene 14 exon splicing mutation in peripheral blood circulation tumor RNA is specifically as follows:

1. the composition of the kit is shown in Table 2.

TABLE 2 composition of kit

Wherein the reagents in the above table were formulated according to the contents of table 3:

TABLE 3 preparation of reagents

2. Storage conditions and expiration dates

The kit is stored at the temperature of minus 20 plus or minus 5 ℃ and the effective period is 12 months; the effective period after unsealing is 6 months; repeatedly freezing and thawing for 3 times.

3. Suitable instrument

ABI 7500 fluorescent PCR instrument

4. Sample requirement

1. Sample type: a plasma sample.

2. And (3) sample collection:

blood was collected 10mL using K2EDTA or free nucleic acid blood collection tubes, as recommended by the manufacturer, and the blood sample should be immediately processed. Before plasma preparation, K2EDTA blood collection tube is kept at 2-8deg.C for no more than 24 hr without freezing blood sample. The free nucleic acid sample tube can be stored at 15-25deg.C for no more than 72 hr before plasma preparation, without freezing blood sample.

3. Preparation and preservation of plasma:

putting the blood collection tube into a centrifugal machine for centrifugation, wherein the rotation speed is 1350+/-150 rcf, and the centrifugation is carried out for 12 minutes (the braking function of the centrifugal machine is forbidden to be used so as to prevent the damage of a blood cell layer); the blood collection tube is taken out from the centrifuge, the blood plasma is transferred to a centrifuge tube made of polypropylene and 15mL with a conical bottom by a new disposable pipette, and the blood collection tube is centrifuged again for 12 minutes at 1350+/-150 rcf. 4mL of plasma was added to a new centrifuge tube using a new disposable pipette or a serum pipette, and the sample number was labeled. The plasma sample can be used for detection immediately, or can be stored at 2-8deg.C (not more than 24 h), and can be stored for one month at 15-25deg.C and for six months at below-70deg.C. The rpm was converted to rcf by reference to the centrifuge operating manual.

4. Sample usage: 4mL.

5. Inspection method

1. Reagent preparation

1.1 And taking out the PCR reaction liquid from the kit, thawing at room temperature, gently shaking and uniformly mixing after full thawing, and performing short-term centrifugation for later use.

1.2 According to the number (n) of samples to be detected, the required reaction number is calculated to be n+3 (negative quality control, positive quality control and no template control), and each reaction is divided into 15 mu l of PCR reaction liquid.

2. Sample processing

2.1 Thawing of plasma

In the case of frozen plasma samples, which are thawed for about 30 minutes at room temperature (15-30 ℃), the sample must undergo a lysis procedure within 60 minutes of thawing.

2.2 Nucleic acid extraction

The free RNA extraction or purification kit was operated for plasma free RNA extraction with reference to commercial kit instructions purchased.

3. Sample addition

Sample adding: and respectively adding RNA to be tested into the PCR reaction tubes prepared with the reagents. Each PCR reaction was supplemented with 10. Mu.L cfRNA, as shown in Table 4, and after closing the tube cap, the pellet was centrifuged at a low speed. Note that: the sealed PCR plate can be left at 2-8℃for up to 4 hours.

TABLE 4 RET PCR Mix and sample mixing Table

4. PCR amplification

4.1 Sample setting: sample numbers are set according to sample types, and the 8-row sample adding layout of the PCR instrument is shown in Table 5. In the table, PC represents Positive Control, NC represents Negative Control, NTC represents no-template Control (No Template Control), and S represents sample.

Table 5 8 in-line sample loading layout table

4.2 Fluorescence channel selection: the MET gene selected FAM channel per sample, the ACTB gene selected JOE channel per sample, and the reference fluorescence (Passive Reference) was set to none.

4.3 The reaction conditions were set (reaction volume was set to 25. Mu.L) as shown in Table 6.

TABLE 6 reaction conditions Table

X is the fluorescence detection time.

4.4 And saving the file and running the program.

5. Analysis of results

Automatically storing the result after the reaction is finished, automatically analyzing the result by using instrument matched software, adjusting the Start value, end value and Threshold value of Baserine according to the analyzed image (a user can adjust the Start value at 2-8 and the End value at 10-20 according to actual conditions), setting a fluorescence Threshold (Threshold) to be just higher than the highest point of an amplification curve (an irregular noise line) of a negative quality control product by a Threshold line, displaying the Ct value as undet), and clicking Analysis to automatically obtain the Analysis result.

6. The results are shown in Table 7.

Table 7 results determination table

6. Interpretation of test results

1. Experiment quality judgment: if FAM channel detection in the negative quality control NC is not amplified (not in a typical S-shaped curve, note that the typical S-shaped curve is sequentially expressed as an exponential phase, a straight line phase and a plateau phase) or has no Ct value, JOE channel detection is amplified (in the typical S-shaped curve) and the Ct value is less than or equal to 35; the FAM and JOE channels in the MET14 positive quality control product are amplified (in a typical S-shaped curve) and the Ct value is less than or equal to 35, so that the analysis can be continued; otherwise, the experiment is considered invalid and repeated.

2. Judging the detection condition of an internal reference gene (ACTB) in a sample to be detected: if the internal reference gene detection (JOE channel) is amplified and the Ct value is less than or equal to 35, the analysis can be continued; if the Ct value is large or no amplification is performed, the RNA sample is considered to be too low in concentration or degraded, or may contain PCR inhibitors therein, and RNA detection needs to be re-extracted.

3. Judging the jump mutation condition of the MET14 gene in the sample to be detected: if MET14 locus detection (FAM channel) in the sample is amplified and the Ct value is less than or equal to 39, judging that the detection result of the MET14 gene jump mutation of the sample is positive; if Ct value is >39 or no amplification, then the detection result of the sample MET14 gene jump mutation is negative.

Based on the above flow, a detection system for detecting MET gene 14 exon splicing mutation in peripheral blood circulation tumor RNA is designed, and comprises an acquisition module, an extraction module, an amplification module, a fluorescence detection module and a judgment module;

the collection module is used for collecting a plasma sample;

an extraction module for extracting a free RNA sample from a plasma sample;

the amplification module is used for carrying out PCR amplification on the free RNA sample by adopting a primer probe group or a kit;

the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;

and the analysis module is used for analyzing according to the fluorescence detection result.

Example 2

Extracting cfDNA in a sample to be detected:

13 patients with non-small cell lung cancer are selected, 10mL of blood samples of the patients are taken, and 4mL of plasma samples are separated from the blood samples for free cfRNA extraction. After the RT-qPCR amplification reaction is performed, the fluorescence threshold line can be manually adjusted. Based on the adjusted threshold line, it is determined whether the Ct value of MET E14 and the control gene ACTB is contained in the plasma sample of the subject. The results show that the detection of the internal reference gene (JOE channel) of 3 patients is amplified, the Ct value is less than or equal to 35, the detection of the MET14 site (FAM channel) is amplified, the Ct value is less than or equal to 39, and the detection of the internal reference gene (JOE channel) of the 3 patients is judged to have MET E14 jump deletion in the samples of the 3 patients; detection of 10 patients with amplified reference gene (JOE channel) and Ct value less than or equal to 35 and MET14 site detection (FAM channel) with Ct value >39 or no amplification, was determined that MET E14 jump deletion was not present in samples of these 10 patients. Ct values for MET E14 and ACTB for these 13 patients are shown in table 8.

TABLE 8 Ct values Table of MET E14 and ACTB for 13 patients

The kit comprises:

1) The detection is carried out by adopting a circulating tumor RNA (ctRNA) sample, so that the dynamic monitoring and management of the curative effect can be carried out on an advanced cancer patient incapable of obtaining a tissue sample by ctRNA, and the optimization of the treatment scheme is of great significance in relieving the clinical symptoms of the advanced cancer patient, prolonging the life cycle and improving the life quality.

2) The system is completed in one tube by adopting reverse transcription and qPCR reaction, and no additional tube opening/pipetting operation is needed, so that the detection flux is greatly improved, and the pollution risk is reduced. The kit is introduced with dUTP/UDG anti-pollution system, and the detection sensitivity of the kit can reach 0.1 pg total RNA or RNA templates with copy number less than 10.

3) The probe adopts Taqman-MGB probe design, improves the signal to noise ratio (because the quenching group at the 3' end of the probe is a fluorescent group which does not emit light), and has better detection sensitivity compared with the position of the reporter gene in space.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be understood that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A primer probe group for detecting MET gene 14 exon splicing mutation in peripheral blood circulation tumor RNA is characterized by comprising a primer and a probe with nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 3.

2. The primer probe set for detecting splicing mutation of MET gene 14 exon in peripheral blood circulating tumor RNA according to claim 1, further comprising a primer and a probe having nucleotide sequences shown as SEQ ID NO.4-SEQ ID NO. 6.

3. A kit for detecting a MET gene 14 exon splice mutation in peripheral blood circulation tumor RNA, comprising the primer probe set of claim 1 or 2 for detecting a MET gene 14 exon splice mutation in peripheral blood circulation tumor RNA.

4. The kit according to claim 3, comprising 2 parts by volume of Buffer SG 60, 4 parts by volume of 1M trehalose, 0.6 parts by volume of DMSO, 0.2 parts by volume of 10 mg/mL BSA, 0.16 parts by volume of dntps, 0.1 parts by volume of dUTP, 0.16 parts by volume of ROX, 0.34 parts by volume of 0.1M MgCl2, 0.5 parts by volume of enhancement, 0.4 parts by volume of Taq, 0.8 parts by volume of reverse transcriptase, 0.5 parts by volume of Heat-enzyme nucleotide-DNA glycase, 0.24 parts by volume of nuclease-free water, 1 part by volume of primer of nucleotide sequence shown in SEQ ID No.1, 1 part by volume of primer of nucleotide sequence shown in SEQ ID No.2 and 0.5 parts by volume of probe of nucleotide sequence shown in SEQ ID No. 3;

or, 2 parts by volume of Buffer SG 6, 4 parts by volume of 1M trehalose, 0.6 parts by volume of DMSO, 0.2 parts by volume of 10 mg/mL BSA, 0.16 parts by volume of dNTPs, 0.1 parts by volume of dUTP, 0.16 parts by volume of ROX, 0.34 parts by volume of 0.1M MgCl2, 0.5 parts by volume of an Enhancer, 0.4 parts by volume of Taq, 0.8 parts by volume of reverse transcriptase, 0.5 parts by volume of Heat-enzyme Uracil-DNA Glycosylase, 0.24 parts by volume of nuclease-free water, 1 part by volume of a primer of a nucleotide sequence shown as SEQ ID NO.4, 1 part by volume of a primer of a nucleotide sequence shown as SEQ ID NO.5 and 0.5 parts by volume of a probe of a nucleotide sequence shown as SEQ ID NO.6 are also included.

5. Use of the primer probe set for detecting the splicing mutation of the exon 14 of MET gene in peripheral blood circulating tumor RNA according to claim 1 or 2 for preparing a reagent for detecting the splicing mutation of the exon 14 of MET gene.

6. The use of the kit for detecting a MET gene 14 exon splice mutation in peripheral blood circulating tumor RNA according to claim 3 or 4 for the preparation of a MET gene 14 exon splice mutation detection reagent.

7. The detection system for detecting MET gene 14 exon splicing mutation in peripheral blood circulation tumor RNA is characterized by comprising an acquisition module, an extraction module, an amplification module, a fluorescence detection module and a judgment module;

the collection module is used for collecting a plasma sample;

the extraction module is used for extracting a free RNA sample from the plasma sample;

the amplification module is used for carrying out PCR amplification on the free RNA sample by adopting the primer probe set as claimed in claim 1 or 2 or the kit as claimed in claim 3 or 4;

the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;

and the analysis module is used for analyzing according to the fluorescence detection result.

8. The detection system of claim 7, wherein the reaction conditions for the PCR amplification in the amplification module are: reverse transcription is carried out for 20min at 55 ℃; activating at 95 ℃ for 5min; cycling for 45 times at 95 ℃, 15s,65 ℃, 5s,56 ℃ and 35 s; kept at 25℃for 10s.

9. The detection system according to claim 7, wherein in the analysis module, if the internal reference gene is amplified and the Ct value is less than or equal to 35, the MET14 site is amplified and the Ct value is less than or equal to 39, the detection result is that the sample MET14 gene is splice mutated positive; if the internal reference gene is amplified and the Ct value is less than or equal to 35, and the MET14 locus is not amplified or the Ct value is more than 39, the detection result is that the splicing mutation of the MET14 gene of the sample is negative.

10. A detection method for non-disease diagnostic purposes for detecting a MET gene 14 exon splice mutation in peripheral blood circulating tumor RNA, comprising:

collecting a plasma sample;

extracting a free RNA sample from the plasma sample;

PCR amplification of free RNA samples using the primer-probe set of claim 1 or 2 or the kit of claim 3 or 4;

performing fluorescence detection on the amplified sample;

analyzing according to the fluorescence detection result;

if the internal reference gene is amplified and the Ct value is less than or equal to 35, the MET14 locus is amplified and the Ct value is less than or equal to 39, the detection result is that the splicing mutation of the MET14 gene of the sample is positive; if the internal reference gene is amplified and the Ct value is less than or equal to 35, and the MET14 locus is not amplified or the Ct value is more than 39, the detection result is that the splicing mutation of the MET14 gene of the sample is negative.