CN104818320A - Primers, probes, detection system and kit for one time detection of lung cancer multiple genes - Google Patents
Primers, probes, detection system and kit for one time detection of lung cancer multiple genes Download PDFInfo
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Abstract
The present invention discloses primers, probes, a detection system and a kit for one time detection of lung cancer multiple genes, wherein the primers, the probes, and the distribution way for detecting 25 EGFR gene mutations, 7 KRAS gene mutations, 6 BRAF gene mutations, 9 NRAS gene mutations, 5 HER2 gene mutations, 2 PIK3CA gene mutations, 5 fusion genes of ALK5, 13 fusion genes of ROS1, and 6 fusion genes of RET are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, and the corresponding fusion detection reagents and the internal control reagents are filled in the pipes 1-4 of the 12 linking PCR strip; and with the primers, the probes, the detection system and the kit, the one-time detection of the 24 fusions and the 54 mutations of the lung cancer can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on lung cancer patients can be provided for clinician.
Description
Technical field
The invention belongs to biological technical field, relate to the primer of lung cancer multiple gene, probe and detection system particularly.
Background technology
Lung cancer is one of sickness rate and the highest malignant tumour of case fatality rate in malignant tumour, and wherein nonsmall-cell lung cancer (NSCLC) accounts for about 85% of lung cancer.Along with deepening continuously of studying Tumorigenesis and biological behaviour thereof, people more and more gather high to specificity, that untoward reaction is gently feature molecular targeted therapy focus.In recent years, in molecular targeted therapy for lung cancer field, study hotspot mainly concentrates on the target spots such as EGFR, ALK, ROS1, KRAS, BRAF, the sudden change of these genes or Fusion Strain relevant to the curative effect of targeted drug, polygenic joint-detection can further improve prediction prognosis accuracy.But, the sudden change of a sudden change or a fragment gene can only be detected at present mostly at every turn, detect flux low, can only examination one by one, length consuming time.Meanwhile, because patients with terminal is drawn materials more difficult, sample size is less, often can not meet the needs of examination one by one, and there is no clinically can simultaneously, disposablely complete the test kit that this 9 focus genes are detected.
The invention provides the primer for the multiple lung cancer gene of disposable detection of a kind of highly sensitive and high specific, probe, test kit and primer distribution means.Utilize DNA sample the to carry out detection that transgenation and RNA sample carry out gene fusion of this detection kit, specificity and highly sensitive, detected result is reproducible, and introduce detection technique of fluorescence, overcome the problem of normal PCR product carryover contamination, simple and efficient to handle, for the detection of clinical EGFR, ALK, ROS1, RET, KRAS, BRAF, NRAS, HER2, PIK3CA 9 kinds of transgenation/fusions provides a kind of fast and reliable detection method.The method can realize the multiple gene of disposable detection of lung cancer, substantially reduces the time of detection, improves sensitivity and the accuracy rate of lung cancer gene test, thus realizes instructing lung cancer clinical treatment plan to formulate and chemotherapy prognosis.
Summary of the invention
The object of the present invention is to provide a kind of primer, probe for the multiple lung cancer gene of disposable detection, comprise the primer of the focus genes such as EGFR, BRAF, KRAS, ALK and ROS1, probe and distribution means, its sequence is as table 1:
Table 1 primer, probe and sequence number
E-20-M2-P-HEX
K-EX4-P-C-HEX
Another object of the present invention is to provide the test kit detecting multiple lung cancer gene.This test kit comprises two parts: RNA fusion gene detects and DNA detection in Gene Mutation, comprises the isogenic Auele Specific Primer of EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET, novel probe, also comprises PCR damping fluid, UNG enzyme etc.Test kit adopts 12 PCR to react bar design, wherein RNA fusion gene detect by 12 PCR react bars 1.-4. a number pipe form, wherein 1. number pipe is made up of ALK fusion gene detection reagent and internal control detection reagent, and 3. 2. a number pipe is all made up of ROS1 fusion gene detection reagent and internal control detection reagent, and 4. number pipe is made up of RET fusion gene detection reagent and internal control detection reagent.1.-4. number pipe fusion gene detection reagent is by FAM signal designation, internal control detection reagent for monitoring sample rna quality and adding situation, by HEX (VIC) signal designation; DNA detection in Gene Mutation by 12 PCR react bars 5. ~
number pipe composition, wherein 5. ~
fAM and the HEX signal of number pipe indicates the different genes mutational site of EGFR, KRAS, BRAF, NRAS, HER2 and PIK3CA respectively,
number pipe is made up of the detection reagent in the not mutated district of the EGFR that increases, and is used for the extraction quality of Quality Control DNA, also by FAM and HEX signal designation.
Each pattern detection needs 1 12 PCR to react bar.This test kit composition refers to table 2 and table 3.The present invention, by the disposable detection to gene fusion in transgenation in sample DNA and RNA, instructs lung cancer clinical treatment plan to formulate and chemotherapy prognosis.
Table 2 test kit forms
Table 3 LMG 12 PCR reacts bar moiety
All fused type information that table 4 test kit detects
All mutation type information that table 5 test kit detects
The present invention provides a kind of reaction system for disposable detection of lung cancer gene test on the other hand, specific as follows:
1. fusion gene
DEPC H
2O
10 × PCR damping fluid
Wherein, the composition of LMG mixed enzyme A is as table 6:
Table 6 LMG mixed enzyme A forms
| Material | Concentration | Consumption (μ L) |
| Hs-taq enzyme | 5U/μL | 0.25-0.30 |
| UNG enzyme | 1U/μL | 0.02-0.06 |
| Reversed transcriptive enzyme | 0.1-1.0 |
Preferably, LMG mixed enzyme A consists of Hs-taq enzyme 0.26 μ L, UNG enzyme 0.03 μ L, reversed transcriptive enzyme 0.5 μ L;
2. mutator gene
H
2O
5 × PCR damping fluid
Wherein, the composition of LMG mixed enzyme A is as table 7:
Table 7 LMG mixed enzyme B forms
| Material | Concentration | Consumption (μ L) |
| Hs-taq enzyme | 5U/μL | 0.20-0.25 |
| UNG enzyme | 1U/μL | 0.02-0.08 |
Preferably, LMG mixed enzyme B consists of Hs-taq enzyme 0.20 μ L, UNG enzyme 0.05 μ L.
The using method of test kit of the present invention comprises the following steps:
1. extract DNA and RNA detected in sample, comprise DNA and RNA in the tissue such as fresh pathological tissue, paraffin-embedded tissue or paraffin section, whole blood, blood, blood plasma hydrothorax.
2. DNA and RNA is mixed with mixed enzyme according to corresponding ratio respectively, and join 12 PCR reaction bars.
3. judge detected result according to the Ct value of fluorescent PCR amplification instrument display:
The invention has the beneficial effects as follows:
The present invention adopts Auele Specific Primer and novel probe technology, Two Colour Fluorescence Air conduct measurement pattern, eliminate internal control gene, more mutator gene can be examined, contain more polygene, detect ALK altogether, ROS1, RET tri-fusion genes and EGFR, KRAS, NRAS, BRAF, HER2, PIK3CA six mutator genes, amount to 24 kinds and merge and 54 kinds of sudden changes.This method: (1) establishes PCR in real time system, can disposable detection of lung cancer multiple gene, comprise EGFR gene 25 kinds sudden change, KRAS gene 7 kinds sudden change, NRAS 9 kinds and BRAF 6 kinds of transgenations, HER25 kind are suddenlyd change, PIK3CA 2 kinds sudden change and ALK 5 kinds of fusion genes, ROS113 kind fusion gene and RET 6 kinds of fusion genes (see table 4 and table 5); Tumor-targeting drug treatment can be selected to provide reference to patients with lung cancer for clinician.According to the incidence of each transgenation and each gene fusion, the detection site that this test kit comprises can cover the Patients with Non-small-cell Lung of about 70%.
The present invention is according to many-sided composite factors such as influencing each other between the detection complexity of detected multiple sudden change/position of fusion, primer, competitions, 115 sequences (comprising primer and probe) are divided into groups, be sub-packed in 12 bracings, its advantage is: (1), after grouping, can not interfere with each other with between group primer; (2) upstream shared or the design of downstream primer; Make the mutational site (such as n) identifying as much, primer number used minimum (only need n+1, and usually need 2n), can be cost-saving.(3) adopt 12 bracings, the multiple sudden change/position of fusion of disposable detection, and establish 12 unions and respectively manage general amplification program, make that each pipe amplification program is consistent, operation is consistent, can save the plenty of time.Containing external control detection system in (4) 12 pipes, as the Quality Control to reagent, sample DNA quality, sample rna quality and operation itself, the surveyed area selected is the section that Human genome is guarded relatively, about 100bp, even if DNA/RNA has degraded like this, external control still can react effective DNA and the RNA amount of EGFR/KRAS/BRAF/ALK/ROS1 gene the most truly; (5) highly sensitive, the fusion gene RNA of the gene mutation DNA to 1% and 50 copies can detect; (6) high specificity, the wild type gene group DNA of 10ng and the wild type rna of 5 μ g can not produce non-specific signals; (7) detection time is short, and testing process only needs to complete for 120 minutes; (8) adopt pre-packing, and carry out sealing wax process, the security of transportation can be improved, process of the test can be made easy and simple to handle simultaneously.
Accompanying drawing explanation
Fig. 1 is each part detecting the sensitivity map of plasmid in embodiment 1, wherein:
Fig. 1-1 ALK fusion gene-EML4 exon 13; ALK exon 20
Fig. 1-2 ROS1 fusion gene-CD74 exon 6; ROS1 exon 32
Fig. 1-3 RET fusion gene-KIF5B exon 16; ROS1 exon 12
Fig. 1-4 EGFR genetic mutation-L861Q
Fig. 1-5 KRAS gene mutation G12D
Fig. 1-6 BRAF gene mutation V600E1
Fig. 1-7 HER2 transgenation G776>VC
Fig. 1-8 NRAS transgenation Q61R
Fig. 1-9 PIK3CA-M1 transgenation H1047R
Fig. 2 is each part detecting the PCR figure of plasmid in embodiment 1, wherein:
Fig. 2-1 ALK fusion gene-EML4 exon 13; ALK exon 20
Fig. 2-2 ROS1 fusion gene-CD74 exon 6; ROS1 exon 32
Fig. 2-3 RET fusion gene-KIF5B exon 16; ROS1 exon 12
Fig. 2-4 EGFR genetic mutation-L861Q
Fig. 2-5 KRAS gene mutation G12D
Fig. 2-6 BRAF gene mutation V600E1
Fig. 2-7 HER2 transgenation G776>VC
Fig. 2-8 NRAS transgenation Q61R
Fig. 2-9 PIK3CA transgenation H1047R
Fig. 3 is a part of the positive PCR figure of clinical sample detected result in embodiment 2, wherein:
Fig. 3-1 sample 1 HER2 transgenation
Fig. 3-2 sample 2 A, ALK fusion gene; B, ROS1 fusion gene
Fig. 3-3 sample 3 A, ALK fusion gene; B, EGFR genetic mutation
Wherein, the X-coordinate of each figure is cycle number (cycles); Ordinate zou is fluorescent value, Fluorescence (dR).
Embodiment
Below in conjunction with specific embodiment, the present invention is set forth further.Should be understood that these embodiments are only not used in for the present invention to limit the scope of the invention.Unless otherwise defined or described herein, the scientific and technical term described in this patent and those of ordinary skill in the art understand there is identical implication.
Embodiment 1
According to the human EGFR that Cosmic data are announced, KRAS, BRA, ALK and ROS1 is wildtype gene sequence, with EGFR, KRAS, based on the driven nature mutational site of BRA, ALK and ROS1 and position of fusion, design Auele Specific Primer and novel probe (see table 1 and table 8).
The each pipe distribution of table 8
The present embodiment is respectively with EML4 exon 13; ALK exon 20 (ALK fusion gene), CD74 exon 6; ROS1exon 32 (ROS1 fusion gene), KIF5B exon 16; ROS1 exon 12 (RET fusion gene), L861Q (EGFR genetic mutation), G12D (KRAS gene mutation), V600E1 (BRAF gene mutation), G776>VC (HER2 transgenation), Q61R (NRAS transgenation), H1047R (PIK3CA transgenation) set forth fluorescent PCR of the present invention for example and detect above-mentioned lung cancer gene.
Experiment is respectively with the plasmid template of the armoring RNA and each sudden change that contain above-mentioned fusion gene, and its fluorescent PCR detects and comprises the following steps:
(1) plasmid, armoring RNA process and extraction:
The extraction of each plasmid and armoring RNA adopts extraction test kit to extract, and concrete operation steps of extracting presses the operation of test kit specification sheets.DNA and RNA uses UV spectrophotometer measuring DNA and RNA concentration and purity after extracting and terminating immediately.DNA and RNA OD260/OD280 should between 1.8 ~ 2.1; RNA concentration should between 50 ~ 500 ng/ μ L.
(2) pcr amplification reaction system is set up:
By above-mentioned gained template, as the template of real-time fluorescent PCR amplification, carry out pcr amplification according to following amplification system:
1. fusion gene
DEPC H
2O
10 × PCR damping fluid
2. mutator gene
H
2O
5 × PCR damping fluid
Real-time PCR reactions condition is:
First stage: 42 DEG C of 5min, 95 DEG C of 5min, 1 circulation;
Subordinate phase: 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: collect FAM and HEX (or VIC) signal during the phase III 60 DEG C, performs PCR in real time, preserves file.
(3) judgement of detected result: the Ct value according to the display of fluorescent PCR amplification instrument judges detected result.
1. confirm the reference of non-selected correction fluorescence, pressing pipe order selects single detection reaction pipe to analyze successively, select positive control reaction tubes, sample reaction tubes and negative control pipe simultaneously, then user according to the flex point place of practical situation determination amplification curve rise, can obtain the Ct value of each reaction tubes.
2. negative control 1. ~
the FAM signal of number pipe and 5. ~
the HEX signal of number pipe should rise without amplification curve, if the above-mentioned signal of any pipe has amplification curve to rise, then this time experimental result is invalid, advises again detecting; If 1. ~ 4. number pipe HEX signal and
fAM and the HEX signal of number pipe has amplification curve to rise once in a while, and this belongs to normal phenomenon, does not affect the judgement to detected result.
3. positive control 1. ~
fAM, HEX signal of number pipe should all have amplification curve to rise, and Ct value is generally less than 30, but may fluctuate because different threshold value is arranged.
4. sample 1. ~ 4. number pipe ALK, ROS1 and RET gene fusion yin and yang attribute judge:
(1) sample 1. ~ 4. number pipe HEX amplification of signal curve:
If a) three pipe Ct values all≤27, then continue to analyze;
If b) any pipe Ct value > 27, illustrate that RNA may exist fragmentation or degraded or inhibitor or leakage and add, if FAM amplified signal rises and drops on positive region, experimental result is still credible, otherwise advises detection again or detect after again extracting RNA again.
(2) sample 1. ~ 4. number pipe FAM amplification of signal curve:
If a) 1. number pipe FAM signal Ct value < 35, then this sample contains ALK gene and merges, otherwise, do not merge containing ALK gene;
If b) 2. or 3. number pipe FAM signal Ct value < 35, then this sample contains ROS1 gene fusion,
Otherwise, not containing ROS1 gene fusion.
If c) 4. number pipe FAM signal Ct value < 35, then this sample contains RET gene fusion, otherwise, not containing RET gene fusion.
5. sample 5. ~
number pipe transgenation yin and yang attribute judges (as table 9):
(1) sample
number pipe FAM amplification of signal curve:
If a) 20≤
ct value≤26 of number pipe FAM signal, then continue to analyze;
If b)
the Ct value < 20 of number pipe FAM signal, illustrates that the DNA added is excessive, should reduce DNA add-on and test.But jump signal is without rise or drop on negative district, and this experimental result is still credible;
If c)
the Ct value > 26 of number pipe FAM signal, illustrate that the DNA added may exist fragmentation or degraded or inhibitor or leakage and add, rise if jump signal has and drop on strong positive region, experimental result is still credible, otherwise suggestion increases DNA applied sample amount or detects after again extracting DNA again.
(2) sample 5. ~
number pipe FAM and HEX amplification of signal curve: first determine sample 5. ~
number pipe FAM and HEX signal Ct value separately, then determine this sample
the Ct value of number pipe FAM and HEX signal.Because the percentage composition that suddenlys change in sample is different, the sudden change Ct value obtained is also different.According to different sudden change Ct values, pattern detection result be divided into feminine gender, weak sun and by force sun.Concrete judgement is in table 9.
A) when sample sudden change Ct value is more than or equal to negative threshold value, then this sample is feminine gender or the Monitoring lower-cut lower than this test kit;
B) when sample sudden change Ct value is less than negative threshold value, following judgement is carried out:
I. when the sudden change Ct value of certain reaction tubes is less than strong positive threshold value, then this sample is that the sudden change that this reaction tubes is corresponding is positive, i.e. strong sun;
Ii. when the sudden change Ct value of reaction tubes is more than or equal to strong positive threshold value, then the Δ Ct value of this reaction tubes is calculated.If the Δ Ct value of reaction tubes is less than corresponding Δ Ct Cut-off value, then this sample is also that the sudden change that this reaction tubes is corresponding is positive, i.e. weak sun; Otherwise be then feminine gender or the Monitoring lower-cut lower than this test kit.
C) calculating of Δ Ct value: Δ Ct value=Ct value of suddenling change-external control Ct value.Sudden change Ct value refers to the Ct value that sample jump signal (FAM or HEX signal) is corresponding; External control Ct value refers to the Ct value of the external control signal (FAM or HEX signal) that sample is corresponding.
6. sample may simultaneously containing 2 or multiple fusion or mutation type.
Table 9 transgenation result judges
(3) sample 5 ~ No. 11 pipe FAM amplification of signal curves:
First determine sample 5 ~ No. 11 pipe FAM signals Ct value separately, then determine the Ct value of this sample No. 12 pipe FAM signals.Because the percentage composition that suddenlys change in sample is different, the sudden change Ct value obtained is also different.According to different sudden change Ct values, pattern detection result be divided into feminine gender, weak sun and by force sun.Concrete judgement is in table 9.
Sensitivity analysis: concentration quantitatively of learning from else's experience is the sample RNA/DNA of 1000 copies, and carry out 10 times of dilutions, then detect 3 concentration respectively, every secondary response adds RNA/5 μ L DNA.Result shows the highly sensitive of fluorescence PCR method of the present invention, and 10 copy DNA genomes can detect, and partial results is as Fig. 1.
Detected result shows, detection system of the present invention can disposable accurate detection of lung cancer multiple gene, and the sensitivity of detection can reach 5-10 copy.
Embodiment 2
Use the present invention to detect clinical sample, multiple lung cancer detection in Gene Mutation is carried out to 1100 routine clinical lung cancer paraffin-embedded tissue samples year October in December, 2013 ~ 2014.
One, the extraction of sample DNA and RNA is detected: the extraction of DNA and RNA can use FFPE sample DNA/RNA separating kit (tall building, Fujian food medicine prison No. 1400070th, tool (standard) word 2013) altogether of Xiamen Amoydx Bio-Pharmaceutical Technology Co., Ltd., Cat No.ADx-FF03 or QIAGEN paraffin organization DNA extraction kit, Cat NO.56404; Operate according to the explanation of extracting test kit.DNA and RNA uses UV spectrophotometer measuring DNA and RNA concentration and purity after extracting and terminating immediately.DNA and RNA OD260/OD280 should between 1.8 ~ 2.1; RNA concentration should between 50 ~ 500 ng/ μ L.DNA and RNA of said extracted is used as the fluorescent PCR amplification template of lung cancer gene.
Two, by above-mentioned carried each DNA and RNA, pcr amplification is carried out according to following amplification system respectively:
1. fusion gene
DEPC H
2O
10 × PCR damping fluid
2. mutator gene
H
2O
5 × PCR damping fluid
Real-time PCR reactions condition is:
First stage: 42 DEG C of 5min, 95 DEG C of 5min, 1 circulation;
Subordinate phase: 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: collect FAM and HEX (or VIC) signal during the phase III 60 DEG C, performs PCR in real time, preserves file
Three, the judgement of detected result: judge detected result (determination methods is with reference to embodiment 1) according to the Ct value of fluorescent PCR amplification instrument display.
In the clinical sample of 1100 detected examples, detect 720 routine saltant types altogether, its total mutation rate reaches 65.5%.Wherein, the ratio detecting EGFR sudden change simultaneously and ALK fusion reaches 2.1%, and the ratio that ALK merges and ROS1 merges simultaneously is 0.3%.In addition, adopt direct sequencing to carry out the sudden change example comparison and detection of 100 examples, result shows that the coincidence rate of system of the present invention and direct sequencing reaches 100%, further demonstrates the accuracy that system of the present invention detects, the results are shown in Table 2.The present invention can detection of lung cancer multiple gene, and fast easy to detect, accuracy is high, can meet lung cancer Gene Detecting.The coincidence rate of this fluorescence PCR method and traditional sequencing methods result is 100%, and fluorescent PCR sensitivity and selective enumeration method ability are higher than traditional sequencing methods.
Table 9 detected result of the present invention compares with direct sequencing
Claims (5)
1. the location mode of primer and probe in a test kit, it is characterized in that: adopt 12 unions or 8 unions, by primer and probe sequence grouping, be sub-packed in 12 unions or 8 unions, wherein, (1) can not cause interfering with each other of primer dimer aspect with between group primer; (2) upstream or downstream primer is shared with group primer; (3) primer of all groups is consistent with probe amplification program, operation is consistent; (4), in the different probe organized, at least there is dichromatic dye, to provide at least double-colored sense channel.
2. the primer of disposable detection of lung cancer multiple gene and probe, comprise following 115 sequences, and described sequence is divided into 12 groups, specific as follows:
3. the reaction system of disposable detection of lung cancer gene test, comprises
1. fusion gene
DEPC H
2O
10 × PCR damping fluid
Wherein, LMG mixed enzyme A's is composed as follows:
2. mutator gene
H
2O
5 × PCR damping fluid
Wherein, LMG mixed enzyme B's is composed as follows:
Wherein, described primer and probe are as shown in claim 1.
4. the reaction system of disposable detection of lung cancer gene test as claimed in claim 3, is characterized in that, the LMG mixed enzyme A added consists of Hs-taq enzyme 0.26 μ L, UNG enzyme 0.03 μ L, reversed transcriptive enzyme 0.5 μ L; LMG mixed enzyme B consists of Hs-taq enzyme 0.20 μ L, UNG enzyme 0.05 μ L.
5. a test kit, comprises 12 groups of sequences according to claim 2, and these 12 groups of sequences are respectively charged in 12 unions.
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