CN104818320B - Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene - Google Patents
Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene Download PDFInfo
- Publication number
- CN104818320B CN104818320B CN201410727044.4A CN201410727044A CN104818320B CN 104818320 B CN104818320 B CN 104818320B CN 201410727044 A CN201410727044 A CN 201410727044A CN 104818320 B CN104818320 B CN 104818320B
- Authority
- CN
- China
- Prior art keywords
- kinds
- detection
- lung cancer
- gene
- enzymes
- 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.)
- Active
Links
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of primer, probe, detection architecture and kit for detecting lung cancer multiple gene, including for detecting the 25 kinds of mutation of lung cancer EGFR gene, the 7 kinds of mutation of KRAS genes and 5 kinds of 6 kinds of gene mutations of BRAF, 9 kinds of gene mutations of NRAS, HER2 gene mutations, the primer, probe and its method for salary distribution of 5 kinds of fusions of 2 kinds of gene mutations of PIK3CA and ALK, 13 kinds of fusions of ROS1 and 6 kinds of fusions of RET.The detection kit of the present invention detects the multiple gene of a sample using 12 PCR reaction bar designs, each 12 PCR bar, and 1 No. 4 pipes of 12 bracings are built with corresponding fusion detection reagent and internal control reagent.The present invention can realize the 24 kinds of fusions and 54 kinds of mutation of disposable detection lung cancer, substantially reduce the time of detection, and high sensitivity, high specificity is simple and efficient to handle, tumor-targeting drug treatment can be selected to provide reference patients with lung cancer for clinician.
Description
Technical field
The invention belongs to biological technical field, more particularly to the primer, probe and detection architecture of lung cancer multiple gene.
Background technology
Lung cancer is one of the incidence of disease and case fatality rate highest malignant tumour, wherein non-small cell lung cancer in malignant tumour
(NSCLC) 85% of lung cancer or so is accounted for.With being deepened continuously to Tumorigenesis and its biological behaviour research, Ren Menyue
Come more molecular targeted therapies focus gathered to specific high, adverse reaction with the characteristics of light.In recent years, in lung cancer molecular target
To therapy field, study hotspot is concentrated mainly on the target spots such as EGFR, ALK, ROS1, KRAS, BRAF, the mutation of these genes or
The effect of Fusion Strain is to targeted drug is related, and polygenic joint-detection can further improve the accuracy of prediction prognosis.So
And at present mostly can only detect every time one mutation or one section of gene mutation, detection flux it is low, can only examination one by one, take
It is long.Simultaneously as patients with terminal materials are more difficult, sample size is less, tends not to the needs for meeting examination one by one, and clinically still
It can not simultaneously, disposably complete the kit detected to this 9 focus genes.
The present invention provides the primer for being used to disposably detect multiple lung cancer gene of a kind of high sensitivity and high specific, visited
Pin, kit and the primer method of salary distribution.The detection kit carries out gene mutation using DNA sample and RNA samples carry out gene
The detection of fusion, specificity and high sensitivity, testing result is reproducible, and introduces detection technique of fluorescence, overcomes biography
Unite PCR primer carryover contamination the problem of, it is simple and efficient to handle, be clinical EGFR, ALK, ROS1, RET, KRAS, BRAF, NRAS,
The detection of 9 kinds of gene mutation/fusions of HER2, PIK3CA provides a kind of fast and reliable detection method.This method can be realized
The multiple gene of disposable detection lung cancer, the time of detection is substantially reduced, improve sensitivity and the standard of lung cancer genetic test
True rate, the formulation of lung cancer clinical therapeutic scheme and chemotherapy prognosis are instructed so as to realize.
The content of the invention
It is an object of the invention to provide a kind of primer, probe for being used to disposably detect multiple lung cancer gene, including
Primer, probe and the method for salary distribution of the focus gene such as EGFR, BRAF, KRAS, ALK and ROS1, its sequence such as table 1：
The primer of table 1, probe and sequence number
E-20-M2-P-HEX
K-EX4-P-C-HEX
Another object of the present invention is to provide to detect the kit of multiple lung cancer gene.This kit includes two portions
Point：RNA fusions detect and DNA detection in Gene Mutation, include the bases such as EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET
Specific primer, the novel probe of cause, in addition to PCR buffer solutions, UNG enzymes etc..Kit is designed using 12 PCR reaction bars,
Wherein RNA fusions detection by 12 PCR react bar 1. -4. a number pipe form, wherein 1. a number pipe is detected by ALK fusion gene
Reagent and internal control detection reagent composition, 2. and 3. a number pipe is all made up of ROS1 fusions detection reagent and internal control detection reagent,
4. number pipe is made up of RET fusions detection reagent and internal control detection reagent.1. -4. number pipe fusion detection reagent is by FAM
Signal designation, internal control detection reagent is used to monitor sample rna quality and adds situation, by HEX (VIC) signal designation；DNA genes
Abrupt climatic change by 12 PCR react bar 5.~Number pipe composition, wherein 5.~FAM the and HEX signals of number pipe indicate respectively
EGFR, KRAS, BRAF, NRAS, HER2 and PIK3CA different genes mutational site,Number pipe is by expanding the not mutated areas of EGFR
Detection reagent forms, for Quality Control DNA extraction quality, also by FAM and HEX signal designations.
Each pattern detection needs 1 12 PCR reaction bar.This kit forms refers to table 2 and table 3.The present invention passes through
Disposably to the detection of Gene Fusion in gene mutation in sample DNA and RNA, the formulation of lung cancer clinical therapeutic scheme and chemotherapy are instructed
Prognosis.
The kit forms of table 2
The PCR of 3 LMG of table 12 react bar constituent
All fused type information of the kit of table 4 detection
All mutation type information of the kit of table 5 detection
Another aspect of the present invention provides a kind of reaction system for being used to disposably detect lung cancer genetic test, specific as follows：
1. fusion
DEPC H2O
10 × PCR buffer solutions
Wherein, LMG mixed enzymes A composition such as table 6：
The LMG mixed enzymes A of table 6 is formed
| Material | Concentration | Dosage (μ L) |
| Hs-taq enzymes | 5U/μL | 0.25-0.30 |
| UNG enzymes | 1U/μL | 0.02-0.06 |
| Reverse transcriptase | 0.1-1.0 |
Preferably, LMG mixed enzymes A composition is 0.26 μ L, UNG enzyme of Hs-taq enzymes 0.03 μ L, the μ L of reverse transcriptase 0.5；
2. mutator
H2O
5 × PCR buffer solutions
Wherein, LMG mixed enzymes A composition such as table 7：
The LMG mixed enzymes B of table 7 is formed
| Material | Concentration | Dosage (μ L) |
| Hs-taq enzymes | 5U/μL | 0.20-0.25 |
| UNG enzymes | 1U/μL | 0.02-0.08 |
Preferably, LMG mixed enzymes B composition is the μ L of 0.20 μ L, UNG enzyme of Hs-taq enzymes 0.05.
The application method of kit of the present invention comprises the following steps：
1. DNA and RNA in extraction detection sample, including it is fresh pathological tissue, paraffin-embedded tissue or paraffin section, complete
DNA and RNA in the tissue such as blood, blood, blood plasma pleural effusion.
2. DNA and RNA are mixed according to corresponding ratio with mixed enzyme respectively, and it is added to 12 PCR reaction bars.
3. the Ct values shown according to fluorescent PCR amplification instrument judge testing result：
The beneficial effects of the invention are as follows：
The present invention uses specific primer and novel probe technology, Two Colour Fluorescence Air conduct measurement pattern, eliminates internal control base
Cause, more mutators can be examined, cover more polygenes, detect ALK altogether, tri- fusions of ROS1, RET and EGFR,
Six mutators of KRAS, NRAS, BRAF, HER2, PIK3CA, altogether 24 kinds of fusions and 54 kinds of mutation.The method：(1) establish
Real-time PCR system, can disposably detect lung cancer multiple gene, including 25 kinds of mutation of EGFR gene, 7 kinds of KRAS genes are prominent
Change, 9 kinds of NRAS and 6 kinds of gene mutations of BRAF, the mutation of HER25 kinds, 2 kinds of mutation of PIK3CA and 5 kinds of fusions of ALK,
6 kinds of fusions of ROS113 kinds fusion and RET (be shown in Table 4 and table 5)；Can be that clinician selects to swell to patients with lung cancer
The treatment of knurl targeted drug provides reference.According to each gene mutation and the incidence of each Gene Fusion, detection that this kit includes
Site can cover about 70% Patients with Non-small-cell Lung.
The present invention according to influencing each other between the detection complexities of the multiple mutation/position of fusion detected, primer,
Many-sided composite factors such as competition, 115 sequences (including primer and probe) are grouped, are sub-packed in 12 bracings, its advantage exists
In：(1) after packet, will not be interfered with each other between group primer；(2) design of shared upstream or anti-sense primer；Make
Mutational site (such as n) as much must be identified, primer bar number used is minimum (only need n+1, and usually require 2n),
Can be with cost-effective.(3) 12 bracings are used, disposably detect multiple mutation/position of fusion, and establish 12 unions respectively manage it is general
Amplification program, make that each pipe amplification program is consistent, operation is consistent, can save the plenty of time.Detected in (4) 12 pipes containing external control
System, as to reagent, sample DNA quality, sample rna quality and the Quality Control of operation itself, the detection zone of selection is people
The relatively conservative section of genoid, about 100bp, even if so DNA/RNA has degraded, external control remains able to most truly react
The effective DNA and RNA amounts of EGFR/KRAS/BRAF/ALK/ROS1 genes；(5) high sensitivity, to 1% gene mutation DNA and
The fusion RNA of 50 copies can be detected；(6) high specificity, 10ng wild type gene group DNA and 5 μ g wild type rna
Non-specific signals will not be produced；(7) detection time is short, and detection process only needs to complete for 120 minutes；(8) using pre- point
Dress, and sealing wax processing is carried out, the security of transportation can be improved, while experiment process operation can be made easy.
Brief description of the drawings
Fig. 1 is a part for the sensitivity map of each detection plasmid in embodiment 1, wherein：
Fig. 1-1 ALK fusion genes-EML4 exon 13；ALK exon 20
Fig. 1-2 ROS1 fusions-CD74 exon 6；ROS1 exon 32
Fig. 1-3 RET fusions-KIF5B exon 16；ROS1 exon 12
Fig. 1-4 EGFR genetic mutations-L861Q
Fig. 1-5 KRAS gene mutations G12D
Fig. 1-6 BRAF gene mutations V600E1
Fig. 1-7 HER2 gene mutations G776>VC
Fig. 1-8 NRAS gene mutations Q61R
Fig. 1-9 PIK3CA-M1 gene mutations H1047R
Fig. 2 is a part for the PCR figures of each detection plasmid in embodiment 1, wherein：
Fig. 2-1 ALK fusion genes-EML4 exon 13；ALK exon 20
Fig. 2-2 ROS1 fusions-CD74 exon 6；ROS1 exon 32
Fig. 2-3 RET fusions-KIF5B exon 16；ROS1 exon 12
Fig. 2-4 EGFR genetic mutations-L861Q
Fig. 2-5 KRAS gene mutations G12D
Fig. 2-6 BRAF gene mutations V600E1
Fig. 2-7 HER2 gene mutations G776>VC
Fig. 2-8 NRAS gene mutations Q61R
Fig. 2-9 PIK3CA gene mutations H1047R
Fig. 3 is a part for clinical sample testing result positive PCR figures in embodiment 2, wherein：
The HER2 gene mutations of Fig. 3-1 samples 1
The A of Fig. 3-2 samples 2, ALK fusion gene；B, ROS1 fusions
The A of Fig. 3-3 samples 3, ALK fusion gene；B, EGFR genetic mutation
Wherein, the abscissa of each figure is period (cycles)；Ordinate is fluorescent value, Fluorescence
(dR)。
Embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments be only used for the present invention and
It is not used in limitation the scope of the present invention.Unless otherwise defined or described herein, the scientific and technical term described in this patent and this area are general
Logical technical staff is understood with identical implication.
Embodiment 1
The human EGFR announced according to Cosmic data, KRAS, BRA, ALK and ROS1 are wildtype gene sequence, with
Based on EGFR, KRAS, BRA, ALK and ROS1 driven nature mutational site and position of fusion, to design specific primer and new
Type probe (be shown in Table 1 and table 8).
8 each pipe of table is distributed
The present embodiment is respectively with EML4 exon 13；ALK exon 20 (ALK fusion gene), CD74 exon 6；
ROS1exon 32 (ROS1 fusions), KIF5B exon 16；ROS1 exon 12 (RET fusions), L861Q (EGFR
Gene mutation), G12D (KRAS gene mutation), V600E1 (BRAF gene mutation), G776>VC (HER2 gene mutations), Q61R
Illustrate that the fluorescent PCR of the present invention detects above-mentioned lung cancer base exemplified by (NRAS gene mutations), H1047R (PIK3CA gene mutations)
Cause.
Experiment is included with the armoring RNA containing above-mentioned fusion and the plasmid template being respectively mutated, the detection of its fluorescent PCR respectively
Following steps：
(1) plasmid, armoring RNA processing and extraction：
Each plasmid and armoring RNA extraction are extracted using extracts kit, and specific operating procedure of extracting presses kit
Specification operates.After DNA and RNA extractions terminate, immediately using UV spectrophotometer measuring DNA and RNA concentration and purity.
DNA and RNA OD260/OD280 should be between 1.8~2.1；RNA concentration should be between 50~500 ng/ μ L.
(2) pcr amplification reaction system is established：
By above-mentioned gained template, the template as real-time fluorescent PCR amplification, enter performing PCR amplification according to following amplification system：
1. fusion
DEPC H2O
10 × PCR buffer solutions
2. mutator
H2O
5 × PCR buffer solutions
Real-time PCR reactions condition is：
First stage：42 DEG C of 5min, 95 DEG C of 5min, 1 circulation；
Second stage：95 DEG C of 25s, 64 DEG C of 20s, 72 DEG C of 20s, 10 circulations；
Phase III：93 DEG C of 25s, 60 DEG C of 35s, 72 DEG C of 20s, 36 circulations；
Signal collection：FAM and HEX (or VIC) signal are collected during 60 DEG C of phase III, performs real-time PCR, preserves file.
(3) judgement of testing result：The Ct values shown according to fluorescent PCR amplification instrument judge testing result.
1. confirming non-selected correction fluorescence reference, single detection reaction tube is selected to be analyzed successively by pipe order, together
When select positive control reaction tube, sample reaction tube and negative control pipe, then user can determine that amplification is bent according to actual conditions
At the flex point that line rises, the Ct values of each reaction tube are obtained.
2. negative control 1.~FAM signals of number pipe and 5.~The HEX signals of number pipe should rise without amplification curve, if
Any one above-mentioned signal of pipe has amplification curve rise, then this time experimental result is invalid, it is proposed that detects again；1. if~4. number pipe
HEX signals andFAM the and HEX signals of number pipe have amplification curve rise once in a while, this category normal phenomenon, do not influence to testing result
Judgement.
3. positive control 1.~FAM, HEX signal of number pipe should all have amplification curve rise, and Ct values are general
Less than 30, but may be fluctuated because different threshold values are set.
4. sample 1.~4. number pipe ALK, ROS1 and RET Gene Fusion negative and positive sex determination：
(1) sample 1.~4. number pipe HEX amplification of signal curves：
A) if three pipe Ct values≤27, continue to analyze；
If b) any one pipe Ct values ＞ 27, illustrate that RNA there may be fragmentation or degraded or inhibitor or leakage adds, if FAM
Amplified signal rises and fallen in positive region, and experimental result is still credible, otherwise suggests detecting again or extracts again after RNA again
Detected.
(2) sample 1.~4. number pipe FAM amplification of signal curves：
A) if 1. number pipe FAM signal Ct values ＜ 35, the sample contain ALK gene fusion, otherwise, melted without ALK gene
Close；
If b) 2. or 3. number pipe FAM signal Ct values ＜ 35, the sample contains ROS1 Gene Fusions,
Otherwise, without ROS1 Gene Fusions.
If c) 4. number pipe FAM signal Ct values ＜ 35, the sample contains RET Gene Fusions, otherwise, melts without RET genes
Close.
5. sample 5.~Number pipe gene mutation negative and positive sex determination (such as table 9)：
(1) sampleNumber pipe FAM amplification of signal curves：
If a) 20≤Ct value≤26 of number pipe FAM signals, then continue to analyze；
If b)The Ct values ＜ 20 of number pipe FAM signals, illustrate that the DNA added is excessive, DNA additions should be reduced and carry out reality again
Test.But for jump signal without rising or falling in negative area, the experimental result is still credible；
If c)The Ct values ＞ 26 of number pipe FAM signals, illustrate that the DNA added there may be fragmentation or degraded or inhibitor
Or leakage adds, if jump signal has rise and fallen in strong positive region, experimental result is still credible, otherwise suggests increase DNA applied sample amounts
Or again extract DNA after detected again.
(2) sample 5.~Number pipe FAM and HEX amplification of signal curve：First determine sample 5.~Number pipe FAM and HEX
The respective Ct values of signal, it is then determined that the sampleThe Ct values of number pipe FAM and HEX signal.Due to being mutated percentage composition in sample
Different, resulting mutation Ct values are also different.According to different mutation Ct values, pattern detection result is divided into the moon
Property, weak positive and strong sun.It is specific to judge to be shown in Table 9.
A) when sample mutation Ct values are more than or equal to negative critical value, then the sample is for feminine gender or less than this kit
Monitoring lower-cut；
B) when sample mutation Ct values are less than negative critical value, following judgement is carried out：
I. when the mutation Ct values of some reaction tube are less than strong positive critical value, then the sample is to be dashed forward corresponding to the reaction tube
Become positive, i.e., strong sun；
Ii. when the mutation Ct values of reaction tube are more than or equal to strong positive critical value, then the Δ Ct values of the reaction tube are calculated.
If the Δ Ct values of reaction tube are less than corresponding Δ Ct Cut-off values, also mutation is positive corresponding to the reaction tube for the sample
Property, i.e., weak sun；It is on the contrary then be the negative or Monitoring lower-cut less than this kit.
C) calculating of Δ Ct values：Δ Ct values=be mutated Ct values-external control Ct values.Mutation Ct values refer to sample jump signal (FAM
Or HEX signals) corresponding to Ct values；External control Ct values refer to the Ct values of external control signal corresponding to sample (FAM or HEX signals).
6. a sample may contain 2 or multiple fusions or mutation type simultaneously.
The gene mutation result judgement of table 9
(3) 5~No. 11 pipe FAM amplification of signal curves of sample：
5~No. 11 respective Ct values of pipe FAM signals of sample are determined first, it is then determined that No. 12 pipe FAM signals of the sample
Ct values.Due to being mutated in sample, percentage composition is different, and resulting mutation Ct values are also different.According to different mutation
Ct values, pattern detection result is divided into negative, weak positive and strong sun.It is specific to judge to be shown in Table 9.
Sensitivity analysis：Concentration is the sample RNA/DNA of 1000 copies after learning from else's experience quantitatively, carries out 10 times of dilutions, then
3 concentration are detected respectively, RNA/5 μ L DNA are added per secondary response.As a result the spirit of the fluorescence PCR method of the present invention is shown
Sensitivity is high, and 10 copy DNA genomes can detect, partial results such as Fig. 1.
Testing result shows that detection architecture of the invention disposably can accurately detect lung cancer multiple gene, the spirit of detection
Sensitivity can reach 5-10 copies.
Embodiment 2
Clinical sample is detected with the present invention, in December, 2013~2014 year October is to 1100 clinical lung cancer FFPEs
Tissue samples carry out multiple lung cancer detection in Gene Mutation.
First, sample DNA and RNA extraction are detected：Xiamen Ai De biological medicine science and technology can be used to have for DNA and RNA extraction
FFPE sample DNAs/RNA of limit company isolates kit (Fujian tall building food medicine prison tool (standard) word 2013 the 1400070th), Cat
No.ADx-FF03 or QIAGEN paraffin organization DNA extraction kits, Cat NO.56404；Enter according to the explanation of extracts kit
Row operation.After DNA and RNA extractions terminate, immediately using UV spectrophotometer measuring DNA and RNA concentration and purity.DNA and
RNA OD260/OD280 should be between 1.8~2.1；RNA concentration should be between 50~500 ng/ μ L.By the DNA and RNA of said extracted
Fluorescent PCR amplification template as lung cancer gene.
2nd, by above-mentioned carried each DNA and RNA, performing PCR amplification is entered according to following amplification system respectively：
1. fusion
DEPC H2O
10 × PCR buffer solutions
2. mutator
H2O
5 × PCR buffer solutions
Real-time PCR reactions condition is：
First stage：42 DEG C of 5min, 95 DEG C of 5min, 1 circulation；
Second stage：95 DEG C of 25s, 64 DEG C of 20s, 72 DEG C of 20s, 10 circulations；
Phase III：93 DEG C of 25s, 60 DEG C of 35s, 72 DEG C of 20s, 36 circulations；
Signal collection：FAM and HEX (or VIC) signal are collected during 60 DEG C of phase III, performs real-time PCR, preserves file
3rd, the judgement of testing result：The Ct values shown according to fluorescent PCR amplification instrument judge testing result, and (determination methods are joined
According to embodiment 1).
In the clinical sample of 1100 detected, 720 saltant types are detected altogether, and its total mutation rate is up to 65.5%.Its
In, the ratio of detection EGFR mutation simultaneously and ALK fusions is up to 2.1%, while the ratio of ALK fusions and ROS1 fusions is 0.3%.
In addition, the mutation example contrasting detection of 100 is carried out using direct sequencing, the results showed that system of the present invention and direct sequencing
Coincidence rate reaches 100%, further demonstrates the accuracy of system detection of the present invention, the results are shown in Table 2.The detectable lung cancer of the present invention
Multiple gene, easy to detect quick, accuracy is high, can meet lung cancer Gene Detecting.The fluorescence PCR method and tradition are sequenced
The coincidence rate of methods and resultses is 100%, and fluorescent PCR sensitivity and selective enumeration method ability are higher than traditional sequencing methods.
9 testing result of the present invention of table is compared with direct sequencing
Claims (4)
1. the disposably primer and probe of detection lung cancer multiple gene, including following 115 sequences, and described sequence is divided into 13
Group, it is specific as follows：
。
2. the disposably reaction system of detection lung cancer genetic test, including
1. fusion
DEPC H2O
10 × PCR buffer solutions
Wherein, LMG mixed enzymes A composition is as follows：
2. mutator
H2O
5 × PCR buffer solutions
Wherein, LMG mixed enzymes B composition is as follows：
Wherein, described primer and probe is as shown in claim 1.
3. the reaction system of disposable detection lung cancer genetic test as claimed in claim 2, it is characterised in that the LMG of addition is mixed
Synthase A composition is μ L of Hs-taq enzymes 0.26, μ L of UNG enzymes 0.03, the μ L of reverse transcriptase 0.5；LMG mixed enzymes B composition is Hs-
μ L of taq enzymes 0.20, the μ L of UNG enzymes 0.05.
4. a kind of kit, including the nucleotides of 13 groups of sequences described in claim 1, this 13 groups of sequences are respectively charged into 12 unions
It is interior, wherein, reaction solution 1.-reaction solutionAnd external control is separately added into the pipe in 12 unions, and internal control add reaction solution 1.-it is anti-
In answering liquid 4..
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410727044.4A CN104818320B (en) | 2014-12-03 | 2014-12-03 | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410727044.4A CN104818320B (en) | 2014-12-03 | 2014-12-03 | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104818320A CN104818320A (en) | 2015-08-05 |
| CN104818320B true CN104818320B (en) | 2018-02-06 |
Family
ID=53728800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410727044.4A Active CN104818320B (en) | 2014-12-03 | 2014-12-03 | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104818320B (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106801104A (en) * | 2015-11-04 | 2017-06-06 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primer, kit and purposes |
| CN105349654B (en) * | 2015-11-23 | 2019-02-12 | 厦门艾德生物医药科技股份有限公司 | A kind of probe for detecting EGFR genetic mutation, primer, detection architecture and kit |
| CN106636442B (en) * | 2017-02-23 | 2020-12-01 | 上海鼎晶生物医药科技股份有限公司 | Human tumor gene variation joint detection kit |
| CN106978497A (en) * | 2017-04-26 | 2017-07-25 | 武汉友芝友医疗科技股份有限公司 | Detection primer, probe and the detection kit of EML4 ALK fusion gene mutations |
| ES2883934T3 (en) * | 2017-05-31 | 2021-12-09 | Roche Diagnostics Gmbh | Multiplex PCR detection of ALK, RET and ROS fusions |
| KR101987065B1 (en) * | 2017-08-07 | 2019-06-10 | 주식회사 싸이토젠 | A method for analyzing eml4-alk gene variance |
| CN107385088A (en) * | 2017-09-04 | 2017-11-24 | 厦门飞朔生物技术有限公司 | A kind of multiple fluorescence PCR detection reagent box for being used to detect mankind's RET Gene Fusions |
| CN109504769B (en) * | 2017-09-15 | 2022-06-21 | 益善生物技术股份有限公司 | Specific primer, liquid phase chip kit and method for detecting HER2 gene mutation |
| CN108085390A (en) * | 2017-12-29 | 2018-05-29 | 上海桐树生物科技有限公司 | Tumour drives detection agent, detection kit and its application of genetic mutation |
| CN110551815A (en) * | 2018-05-30 | 2019-12-10 | 苏州云泰生物医药科技有限公司 | Kit for detecting human Ras gene mutation and using method thereof |
| CN110938680A (en) * | 2019-12-25 | 2020-03-31 | 江苏为真生物医药技术股份有限公司 | Gene variation site detection method for simultaneously detecting DNA and RNA |
| CN110964830A (en) * | 2019-12-31 | 2020-04-07 | 中山大学达安基因股份有限公司 | Kit and method for multiple detection of ROS1 gene mutation |
| CN111172281B (en) * | 2019-12-31 | 2023-10-20 | 广州达安基因股份有限公司 | Kit and method for detecting multiple gene mutations of non-small cell lung cancer |
| CN111235272B (en) * | 2020-01-10 | 2023-07-07 | 厦门艾德生物医药科技股份有限公司 | Composition for once detecting multiple gene mutation of lung cancer and application thereof |
| CN111139301B (en) * | 2020-03-10 | 2020-12-18 | 无锡市第五人民医院 | Breast cancer related gene ERBB2 site g.39397319C > A mutant and application thereof |
| CN112458169A (en) * | 2020-11-25 | 2021-03-09 | 北京科途医学科技有限公司 | Nucleic acid reagent, kit and detection system for HER2 gene mutation detection |
| CN113355423B (en) * | 2021-07-07 | 2022-06-07 | 安徽科技学院 | Primer probe and kit for detecting mutation of EGFR gene L858R and application of primer probe and kit |
| CN114214412B (en) * | 2021-12-28 | 2024-02-23 | 普瑞斯新(上海)生物医疗科技有限公司 | PIK3CA gene and BRAF gene mutation multiplex detection primer probe and kit thereof |
| CN114410791B (en) * | 2022-01-22 | 2024-03-29 | 河南省肿瘤医院 | Method for detecting lung cancer gene fusion based on NanoString platform |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012170715A1 (en) * | 2011-06-07 | 2012-12-13 | Caris Mpi, Inc. | Molecular profiling for cancer |
| WO2014144121A2 (en) * | 2013-03-15 | 2014-09-18 | Life Technologies Corporation | Classification and actionability indices for lung cancer |
-
2014
- 2014-12-03 CN CN201410727044.4A patent/CN104818320B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012170715A1 (en) * | 2011-06-07 | 2012-12-13 | Caris Mpi, Inc. | Molecular profiling for cancer |
| WO2014144121A2 (en) * | 2013-03-15 | 2014-09-18 | Life Technologies Corporation | Classification and actionability indices for lung cancer |
Non-Patent Citations (3)
| Title |
|---|
| A Single-Tube Multiplexed Assay for Detecting ALK，ROS1，and RET Fusions in Lung Cancer;Maruja E. Lira;《The Journal of Molecular Diagnostics》;20140331;第16卷(第2期);229-243 * |
| Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness toepidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI) therapy in never-smokers with lung adenocarcinoma;Ji-Youn Han;《Lung Cancer》;20140429;第85卷(第2期);161-167 * |
| Multiplexed Molecular Profiling of Lung Cancer;Hiroaki Akamatsu;《Journal of Thoracic Oncology》;20140731;第9卷(第7期);1048-1052 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104818320A (en) | 2015-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104818320B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| CN104818318B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| CN104946739B (en) | EGFR genetic mutation detection kit and its application | |
| Xia et al. | Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls | |
| JP7204004B2 (en) | Reagent kit for batch detection of multiple gene mutations in lung cancer | |
| CN105039580A (en) | Human ALK fusion gene detection primer set and detection kit | |
| CN105256021B (en) | The method and its kit of Sensitive Detection human EGFR gene mutations are sequenced based on Sanger | |
| CN104818317B (en) | Primer, probe, detection architecture and the kit of disposable detection intestinal cancer multiple gene mutation | |
| CN106868139A (en) | PCR primer, kit and purposes | |
| CN104789677A (en) | Circulating tumor DNA EGFR detecting technology and reagent kit thereof | |
| CN105838777A (en) | Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology | |
| CN102776286A (en) | Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation | |
| CN107513578A (en) | A kind of nucleic acid Mass Spectrometry detection method early sieved for lung cancer driving gene and tumor susceptibility gene | |
| CN110055312A (en) | For detecting primer, probe and the kit of the mutation of EGFR gene C797S and T790M cis-trans | |
| CN109593847A (en) | Detect primer pair, kit and the method for the site microsatellite NR24 stability | |
| CN113337608B (en) | Combined marker for early diagnosis of liver cancer and application thereof | |
| CN104293932A (en) | Method for detecting HLA-B * 5801 allele based on real-time fluorescence PCR | |
| CN110373454A (en) | A kind of kit and method of joint-detection EGFR genetic mutation | |
| CN106520994B (en) | It is a kind of for detecting the multiple fluorescence PCR detection reagent box of EGFR genetic mutation | |
| CN107641649B (en) | Primer pair, kit and method for detecting stability of NR27 locus of microsatellite | |
| CN105255869B (en) | Application of ABCC4 gene polymorphism site rs3742106 and detection primer and kit thereof | |
| CN109554475A (en) | Gene mutation/fusion combination and kit for Lung neoplasm malignant and benign lesion | |
| CN113122629B (en) | Primer and probe for hairpin amplification blocking method for gene mutation detection and application thereof | |
| CN105838779A (en) | Method for monitoring and controlling drug resistance of gastrointestinal stromal tumor patient to imatinib/sunitinib through ddPCR technology | |
| CN105838781A (en) | Method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: No. 39, Haicang Ding Shan Road, Haicang District, Xiamen, Fujian Patentee after: AMOY DIAGNOSTICS CO., LTD. Address before: 361000 the 5 floor of No. 4 factory, Chuang Chuang center, 289 Weng Jiao Road, Xinyang street, Haicang District, Xiamen, Fujian, China. Patentee before: Amoy Diagnostics Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200810 Address after: Room 201-202, building 3, 138 xinjunhuan Road, Minhang District, Shanghai Patentee after: Shanghai Xiawei Biotechnology Co.,Ltd. Address before: 361000, No. 39, Ding Shan Road, Haicang District, Fujian, Xiamen Patentee before: Xiamen Aide Biomedical Technology Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201216 Address after: No. 39, Haicang Ding Shan Road, Haicang District, Xiamen, Fujian Patentee after: Xiamen Aide Biomedical Technology Co.,Ltd. Address before: Room 201-202, building 3, 138 Xinjun Ring Road, Minhang District, Shanghai Patentee before: Shanghai Xiawei Biotechnology Co.,Ltd. |