CN117144016A - Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA - Google Patents
Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA Download PDFInfo
- Publication number
- CN117144016A CN117144016A CN202311403442.6A CN202311403442A CN117144016A CN 117144016 A CN117144016 A CN 117144016A CN 202311403442 A CN202311403442 A CN 202311403442A CN 117144016 A CN117144016 A CN 117144016A
- Authority
- CN
- China
- Prior art keywords
- volume
- parts
- primer probe
- detection
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 117
- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 230000004927 fusion Effects 0.000 title claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 33
- 210000005259 peripheral blood Anatomy 0.000 title claims abstract description 23
- 239000011886 peripheral blood Substances 0.000 title claims abstract description 23
- 230000017531 blood circulation Effects 0.000 title claims abstract description 17
- 230000003321 amplification Effects 0.000 claims abstract description 29
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 29
- 239000002773 nucleotide Substances 0.000 claims abstract description 22
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 22
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 21
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 238000007781 pre-processing Methods 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000010839 reverse transcription Methods 0.000 claims description 7
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 6
- 102100034343 Integrase Human genes 0.000 claims description 6
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 239000003623 enhancer Substances 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000001351 cycling effect Effects 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 239000002299 complementary DNA Substances 0.000 abstract description 10
- 238000011529 RT qPCR Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 101000579425 Homo sapiens Proto-oncogene tyrosine-protein kinase receptor Ret Proteins 0.000 description 40
- 102100028286 Proto-oncogene tyrosine-protein kinase receptor Ret Human genes 0.000 description 40
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 32
- 101150077555 Ret gene Proteins 0.000 description 15
- 210000002381 plasma Anatomy 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 11
- 239000008280 blood Substances 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 9
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000010257 thawing Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 101150087690 ACTB gene Proteins 0.000 description 3
- 206010064571 Gene mutation Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 108091092259 cell-free RNA Proteins 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of molecular biology, in particular to a primer probe, a kit and a detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA; the nucleotide sequence of the primer probe is shown as SEQ ID NO.1-SEQ ID NO. 6; the detection system comprises an extraction module, an amplification module, a fluorescence detection module and a judgment module, wherein the extraction module is used for extracting sample free RNA, preprocessing, amplifying the preprocessed free RNA by adopting a primer probe or a kit, and the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample and judging according to the result of the fluorescence detection; the invention adopts qRT-PCR for detection, which has higher speed and better sensitivity; and designing a specific primer and a probe for detecting RET cDNA, and designing an internal reference gene, a primer probe and PCR conditions thereof to realize detection of RET fusion genes.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a primer probe, a kit and a detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA.
Background
Transfected proto-oncogene (RET), a transmembrane glycoprotein receptor tyrosine kinase, is encoded by the RET proto-oncogene located on chromosome 10. Consists of three parts, namely an extracellular region, a transmembrane region and an intracellular tyrosine kinase.
RET gene fusion occurs in non-small cell lung cancer patients in China at a rate of 1.4% -2.5%. At present, targeted drugs aiming at RET gene fusion are marketed in batches at home and abroad, and the curative effect is remarkable. Accurate detection of RET gene fusion is a precondition for the implementation of RET inhibitor therapy.
Patients with RET rearrangements have similar clinical characteristics to ALK, ROSI rearranged patients, relatively young (definite age <60 years), mostly non-smokers, mainly in lung adenocarcinoma. In NSCLC, at least 12 fusion RET partner genes (KIF 5B-RET, CCDC6-RET,
NCOA4-RET, MYO5C-RET, EPHA5-RET, TRIM33-RET, CLIP1-RET, ERC1-RET, PICALM-RET, FRMD4A-RET, RUFY2-RET, TRIM 24-RET), with KIF5B-RET fusion being most common, followed by CCDC6-RET and NCOA4-RET.
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 RET fusion gene.
The detection principle is that firstly total RNA in a patient sample is extracted, mRNA in the patient sample is taken as a template, a primer capable of aiming at RET 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 RET cDNA is obtained, and finally whether RET fusion gene cDNA exists or not is detected, so that the mutation condition of RET 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 the form of exon fusion, 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.
The detection method of RET fusion gene mutation comprises second generation sequencing or RT-qPCR (reverse transcription-quantitative polymerase chain reaction) direct detection of mRNA of RET fusion gene or second generation sequencing and detection of RET fusion gene mutation at DNA level. 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, a kit and a detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA, so as to solve the problems of high cost, long period and the like when the existing second-generation sequencing is used for detecting RET fusion gene mutation.
In order to solve the problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer probe for detecting RET fusion genes in peripheral blood circulation tumor RNA, wherein the nucleotide sequence of the primer probe is shown as SEQ ID NO.1-SEQ ID NO. 6.
As an embodiment, the primer probe further comprises a primer probe of an internal reference gene, and the nucleotide sequence of the primer probe of the internal reference gene is shown as SEQ ID NO.7-SEQ ID NO. 10.
In a second aspect, the invention provides a kit for detecting RET fusion genes in peripheral blood circulation tumor RNA, which comprises the primer probe for detecting RET fusion genes in peripheral blood circulation tumor RNA.
As one embodiment, it comprises 2 parts by volume of Buffer SG 60 (10×), 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 dNTP, 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 u-DNA glycylase, 0.24 parts by volume of nuclease-free water, 0.5 parts by volume of a primer probe of nucleotide sequence shown as SEQ ID No.1, 0.5 parts by volume of a primer probe of nucleotide sequence shown as SEQ ID No.2, 1 part by volume of a primer probe of nucleotide sequence shown as SEQ ID No.3, 1 part by volume of a primer probe of nucleotide sequence shown as SEQ ID No.4, 0.5 parts by volume of primer probe of nucleotide sequence shown as SEQ ID No.5, and 0.5 parts by volume of primer probe of nucleotide sequence shown as SEQ ID No. 5;
or, it further comprises 2 parts by volume of Buffer SG 60 (10×), 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 dNTP, 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-DNA glycase, 0.24 parts by volume of nuclease-free water, 0.35 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO.7, 0.35 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO.8, and 0.3 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO. 9.
In a third aspect, the invention provides a detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA, which comprises an extraction module, an amplification module, a fluorescence detection module and a judgment module;
the extraction module is used for extracting sample free RNA and carrying out pretreatment;
the amplification module is used for amplifying the pretreated free RNA by adopting the primer probe or the kit;
the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;
and the judging module is used for judging according to the fluorescence detection result.
As an embodiment, the amplification procedure for the amplification is reverse transcription at 55 ℃ for 20min; activating at 95 ℃ for 5min; cycling for 45 times at 95 ℃, 15s,65 ℃, 5s,56 ℃ and 35 s; kept at 25℃for 10s.
In one embodiment, in the determination module, if the result of the fluorescent detection of the sample is Ct >39 or no amplification and the internal reference group Ct is less than or equal to 35, the sample is negative, and if the result of the fluorescent detection of the sample is Ct less than or equal to 39 and the amplification curve is "S" and the internal reference group Ct is less than or equal to 35, the sample is positive.
In a fourth aspect, the present invention provides a detection method for the purpose of non-disease diagnosis for detecting RET fusion gene in peripheral blood circulation tumor RNA, comprising:
extracting sample free RNA and preprocessing;
amplifying the pretreated free RNA by adopting the primer probe or the kit;
performing fluorescence detection on the amplified sample;
and judging according to the fluorescence detection result.
In a fifth aspect, the invention provides an application of the primer probe for detecting RET fusion genes in peripheral blood circulation tumor RNA or the kit for detecting RET fusion genes in peripheral blood circulation tumor RNA in preparation of RET fusion gene detection reagents.
The invention has the beneficial effects that: the invention provides a primer probe, a kit and a detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA, which are detected by qRT-PCR; the qRT-PCR detection speed is higher, the detection result can be obtained in 2 working days generally, the detection sensitivity is better, and a plurality of samples are detected at one time; and specific primers and probes for detecting RET cDNA are designed aiming at KIF5B-RET, CCDC6-RET and NCOA4-RET fusion sites with higher RET gene fusion frequency, and specific internal reference genes, primers, probes and PCR conditions are combined to detect RET fusion genes, so that a foundation is laid for improving the biological clinical detection level and popularizing and applying.
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 of amplification of a positive plasmid standard of the present invention;
FIG. 2 is a graph showing amplification of a negative plasmid standard of the present invention;
FIG. 3 is a graph showing RET gene positive amplification of a sample according to the present invention;
FIG. 4 is a graph showing RET gene negative amplification of the sample 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: specific primers and probes for detecting RET cDNA are designed aiming at KIF5B-RET, CCDC6-RET and NCOA4-RET fusion sites with higher RET gene fusion frequency, and specific internal reference genes, primers, probes and PCR conditions are combined to detect RET fusion genes.
The technical scheme adopted by the invention is as follows:
1. the method is characterized in that an upstream primer, a downstream primer and a specific probe are designed aiming at RET gene RNA, the primer can specifically amplify a segment of sequence of RET gene, and the probe can specifically bind with RET.
2. 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.
3. The synthesis of the primers and probes is shown in Table 1.
Table 1 primer probe and nucleotide sequence table
The method also comprises the following steps before the step 1: and extracting, purifying and measuring the concentration of free RNA in the sample to be detected.
4、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. The kit is introduced with a dUTP/UDG anti-pollution system. 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.
5. Preparation of negative and positive standard substance
And (3) amplifying gene fragment sequences by PCR according to the kit, artificially synthesizing RET 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.
The specific implementation process of the invention is 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 RET 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; FAM and JOE channel detection in RET positive quality control products are amplified (in a typical S-shaped curve) and Ct value is less than or equal to 35, and then continuous analysis can be performed; 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 the RNA sample may contain PCR inhibitors, and RNA detection needs to be re-extracted.
3. Judging RET gene fusion conditions in a sample to be detected: if RET locus detection (FAM channel) in the sample is amplified and the Ct value is less than or equal to 39, judging that the RET fusion gene detection result of the sample is positive; if Ct value is more than 39 or no amplification is carried out, the detection result of the RET fusion gene of the sample is negative.
Based on the flow, a detection system is designed, and comprises an extraction module, an amplification module, a fluorescence detection module and a judgment module;
the extraction module is used for extracting sample free RNA and carrying out pretreatment;
the amplification module is used for amplifying the pretreated free RNA by adopting the primer probe or the kit;
the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;
and the judging module is used for judging according to the fluorescence detection result.
Example 1
Extracting cfDNA in a sample to be detected:
10mL of blood samples of 15 patients with non-small cell lung cancer verified by second generation sequencing are selected, and 4mL of plasma samples are separated from the blood samples, and then free cfRNA extraction is carried out. 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 RET and the control gene ACTB is contained in the plasma sample of the subject. The results show that RET gene fusion mutation is detected in samples of 5 patients, wherein the detection of reference genes (JOE channels) of the 5 patients is amplified, the Ct value is less than or equal to 35, the detection of RET sites (FAM channels) is amplified, the Ct value is less than or equal to 39; and detecting that 10 patients have amplified reference gene detection (JOE channel) and Ct value less than or equal to 35 and RET site detection (FAM channel) has Ct value more than 39 or no amplification, and judging that RET gene fusion mutation does not occur in samples of the 10 patients. The Ct values of RET and ACTB for these 15 patients are shown in table 8.
TABLE 8 Ct values for RET and ACTB for 15 patients
The amplification curve of the positive plasmid standard is shown in figure 1, the amplification curve of the negative plasmid standard is shown in figure 2, and the amplification curves of the RET gene positive and negative samples are shown in figures 3 and 4 respectively.
The kit 1) adopts a circulating tumor RNA (ctRNA) sample for detection, can dynamically monitor and manage the curative effect of a patient with advanced cancer who cannot acquire a tissue sample through ctRNA, optimizes a treatment scheme, and has important significance for relieving clinical symptoms of the patient with advanced tumor, prolonging the life span 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 for detecting RET fusion gene in peripheral blood circulation tumor RNA is characterized in that the nucleotide sequence of the primer probe is shown as SEQ ID NO.1-SEQ ID NO. 6.
2. The primer probe for detecting RET fusion gene in peripheral blood circulation tumor RNA according to claim 1, further comprising a primer probe for an internal reference gene, wherein the nucleotide sequence of the primer probe for the internal reference gene is shown as SEQ ID NO.7-SEQ ID NO. 9.
3. A kit for detecting a RET fusion gene in peripheral blood circulating tumor RNA, comprising the primer probe for detecting a RET fusion gene in peripheral blood circulating tumor RNA of claim 1 or 2.
4. The kit for detecting RET fusion gene in peripheral blood circulation tumor RNA according to claim 3, wherein the kit comprises 2 parts by volume of Buffer SG 60 (10×), 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-labril-DNA glycylase, 0.24 parts by volume of nuclease-free water, 0.5 parts by volume of a primer probe of a nucleotide sequence shown as SEQ ID NO.1, 0.5 parts by volume of a primer probe of a nucleotide sequence shown as SEQ ID NO.2, 1 parts by volume of a primer probe of a nucleotide sequence shown as SEQ ID NO.3, 0.5 parts by volume of a primer probe of a nucleotide sequence shown as SEQ ID NO. 5;
or, 2 parts by volume of Buffer SG 60 (10×), 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 dNTP, 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 glycase, 0.24 parts by volume of nuclease-free water, 0.35 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO.7, 0.35 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO.8 and 0.3 parts by volume of primer probe of nucleotide sequence shown as SEQ ID NO.9 are also included.
5. The detection system for detecting RET fusion genes in peripheral blood circulation tumor RNA is characterized by comprising an extraction module, an amplification module, a fluorescence detection module and a judgment module;
the extraction module is used for extracting sample free RNA and carrying out pretreatment;
the amplification module is used for amplifying the pretreated free RNA by adopting the primer probe according to claim 1 or 2 or the kit according to claim 3 or 4;
the fluorescence detection module is used for carrying out fluorescence detection on the amplified sample;
and the judging module is used for judging according to the fluorescence detection result.
6. The detection system for detecting RET fusion gene in peripheral blood circulating tumor RNA of claim 5, wherein the amplification procedure of the amplification is 55 ℃ reverse transcription for 20min; activating at 95 ℃ for 5min; cycling for 45 times at 95 ℃, 15s,65 ℃, 5s,56 ℃ and 35 s; kept at 25℃for 10s.
7. The system according to claim 5, wherein in the determination module, the sample is negative if the result Ct of the fluorescent detection of the sample is greater than 39 or no amplification is performed and the internal reference group Ct is less than or equal to 35, and the sample is positive if the result of the fluorescent detection of the sample is less than or equal to 39 and the amplification curve is "S" and the internal reference group Ct is less than or equal to 35.
8. A detection method for the purpose of non-disease diagnosis for detecting RET fusion gene in peripheral blood circulating tumor RNA, comprising:
extracting sample free RNA and preprocessing;
amplifying the pretreated free RNA using the primer probe of claim 1 or 2 or the kit of claim 3 or 4;
performing fluorescence detection on the amplified sample;
and judging according to the fluorescence detection result.
9. The use of the primer probe for detecting RET fusion gene in peripheral blood circulating tumor RNA as claimed in claim 1 or 2 in the preparation of RET fusion gene detection reagent.
10. The use of the kit for detecting RET fusion gene in peripheral blood circulating tumor RNA as claimed in claim 3 or 4 in the preparation of RET fusion gene detection reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311403442.6A CN117144016A (en) | 2023-10-27 | 2023-10-27 | Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311403442.6A CN117144016A (en) | 2023-10-27 | 2023-10-27 | Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117144016A true CN117144016A (en) | 2023-12-01 |
Family
ID=88884605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311403442.6A Pending CN117144016A (en) | 2023-10-27 | 2023-10-27 | Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117144016A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361419A (en) * | 2013-05-08 | 2013-10-23 | 厦门艾德生物医药科技有限公司 | Probe, primer and detection kit used for RET gene fusion detection |
| CA2885722A1 (en) * | 2012-09-25 | 2014-04-03 | Chugai Seiyaku Kabushiki Kaisha | Ret inhibitor |
| CN104745719A (en) * | 2015-04-28 | 2015-07-01 | 上海允英医疗科技有限公司 | Primer, probe and detection reagent kit for detecting RET fusion gene |
| WO2018220004A1 (en) * | 2017-05-31 | 2018-12-06 | Roche Diagnostics Gmbh | Multiplex pcr detection of alk, ret, and ros fusions |
| CN109971861A (en) * | 2019-05-05 | 2019-07-05 | 上海睿璟生物科技有限公司 | CCDC6-RET fusion detection kit |
| CN110592217A (en) * | 2019-10-08 | 2019-12-20 | 湖南艾佳生物科技股份有限公司 | Kit for detecting KRAS gene mutation in free DNA of peripheral blood and application thereof |
-
2023
- 2023-10-27 CN CN202311403442.6A patent/CN117144016A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2885722A1 (en) * | 2012-09-25 | 2014-04-03 | Chugai Seiyaku Kabushiki Kaisha | Ret inhibitor |
| CN103361419A (en) * | 2013-05-08 | 2013-10-23 | 厦门艾德生物医药科技有限公司 | Probe, primer and detection kit used for RET gene fusion detection |
| CN104745719A (en) * | 2015-04-28 | 2015-07-01 | 上海允英医疗科技有限公司 | Primer, probe and detection reagent kit for detecting RET fusion gene |
| WO2018220004A1 (en) * | 2017-05-31 | 2018-12-06 | Roche Diagnostics Gmbh | Multiplex pcr detection of alk, ret, and ros fusions |
| CN109971861A (en) * | 2019-05-05 | 2019-07-05 | 上海睿璟生物科技有限公司 | CCDC6-RET fusion detection kit |
| CN110592217A (en) * | 2019-10-08 | 2019-12-20 | 湖南艾佳生物科技股份有限公司 | Kit for detecting KRAS gene mutation in free DNA of peripheral blood and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 夏连续等: "《鼠疫实验室技术手册》", vol. 1, 北京出版社, pages: 98 - 101 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110527710B (en) | Primer, probe and kit for detecting NTRK gene fusion mutation | |
| CN110938693B (en) | Primer group, kit and method for detecting BRAF gene mutation | |
| CN110055312B (en) | Primer, probe and kit for detecting cis-trans mutation of EGFR gene C797S and T790M | |
| CN111733238B (en) | Primer group for identifying 3 splice bodies of PML-RAR alpha fusion gene and kit and application thereof | |
| CN111334580A (en) | PIK3CA gene mutation detection kit | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| CN111534588A (en) | Kit and method for detecting gene mutation in acute lymphocytic leukemia based on fluorescent quantitative PCR | |
| CN106498028B (en) | Diagnostic method and kit for T790M mutation of EGFR | |
| CN110592217A (en) | Kit for detecting KRAS gene mutation in free DNA of peripheral blood and application thereof | |
| CN112662772A (en) | NPM1 gene mutation detection kit | |
| CN111500728A (en) | Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression | |
| CN108531598B (en) | ROS1 gene fusion detection primer, method and kit | |
| CN111826446A (en) | Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer | |
| CN115851935A (en) | Primer probe set and kit for MET gene exon14 jump mutation detection | |
| CN117144016A (en) | Primer probe, kit and detection system for detecting RET fusion gene in peripheral blood circulation tumor RNA | |
| CN112626217B (en) | Kit for quantitatively detecting piRNA-54265 gene | |
| CN117230204A (en) | Primer probe set, kit and detection system for detecting MET gene 14 exon splicing mutation | |
| CN113881759A (en) | EGFR gene mutation detection kit and detection method thereof | |
| CN111218512A (en) | Breast cancer 21 gene detection kit and application method thereof | |
| CN112795653A (en) | Primer probe set and kit for KRAS gene mutation detection and application thereof | |
| CN110964830A (en) | Kit and method for multiple detection of ROS1 gene mutation | |
| CN111560429A (en) | Application of circRNA marker for diagnosing thalassemia | |
| CN117187395A (en) | Primer probe group, kit and system for quantitative detection of MET gene amplification | |
| CN117187396A (en) | Primer probe group, kit and system for quantitative detection of HER2 gene amplification | |
| CN114438202B (en) | SNORD57 detection kit for screening and diagnosing intestinal polyps, intestinal adenomas and/or intestinal cancers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |