CN109486951B - Lung cancer targeted drug gene detection kit, primers and method - Google Patents
Lung cancer targeted drug gene detection kit, primers and method Download PDFInfo
- Publication number
- CN109486951B CN109486951B CN201811472985.2A CN201811472985A CN109486951B CN 109486951 B CN109486951 B CN 109486951B CN 201811472985 A CN201811472985 A CN 201811472985A CN 109486951 B CN109486951 B CN 109486951B
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- library
- dna
- specific primer
- kit
- 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
Images
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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
-
- 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)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a lung cancer targeted drug gene detection kit, primers and a method. The kit and the specific primer set can quickly, accurately and once detect hundreds of lung cancer targeted drug gene mutation sites, and the uniformity, the repeatability, the library building power, the library sequencing data utilization rate, the detection accuracy and the sensitivity of multiple PCR library building by using the specific primers are ideal, and the kit is suitable for various sample types.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a gene detection kit, primers and a method for a lung cancer targeted drug.
Background
The lung cancer is the main cause of cancer death, and according to statistics, 733.3 ten thousands of lung cancers occur in a national population in 2015, and 610.2 ten thousands of lung cancers are the first death. The genotyping can guide the diagnosis and treatment of lung cancer, and the gene detection guide the treatment of lung cancer becomes a new medical mode at present. For example, the National Comprehensive Cancer Network (NCCN) published guidelines indicate that the detection of genes such as EGFR and ALK is required for the diagnosis and treatment of non-small cell lung cancer; when EGFR is sensitive and mutated, EGFR-TKIs targeted drugs are recommended as first-line drugs; in the presence of mutations such as KRAS, EGFR-TKIs are not recommended. In addition, studies have shown that the overall survival and progression-free survival of lung cancer patients undergoing gene-based therapy is significantly higher than that of patients undergoing conventional therapy. Therefore, a product capable of detecting the hot spot mutation site of the lung cancer targeted drug gene is developed, and the survival time of a lung cancer patient can be improved.
The development of a new generation of high-throughput target sequence capture sequencing technology has been widely applied to various biological and medical research. Compared with probe capture, the capture of the target sequence by using the multiple PCR has the obvious advantages of simple operation, low cost, large enrichment of target DNA sequences in a short time and the like, and greatly shortens the detection process and the detection time. However, in general, the more target regions that are to be captured by multiplex PCR, the lower the specificity, accuracy, and uniformity of amplification, and the greater the difficulty of implementing the multiplex PCR capture technique.
Disclosure of Invention
The invention aims to provide a lung cancer targeted drug gene detection kit, primers and a method.
The technical scheme adopted by the invention is as follows:
the invention provides a gene detection kit for lung cancer targeted medication, which comprises: detecting a specific primer group of the lung cancer targeted drug gene; the specific primer set includes specific primers designed for target regions of genes shown in table 1 of the specification.
Preferably, the specific primer group comprises specific primers shown by SEQ ID NO. 1-SEQ ID NO. 56.
According to the requirement of library construction, the 5' end of each specific primer in the specific primer group is also connected with a universal sequence required by library construction and sequencing. The universal sequence is selected from a common primer, a joint and a specific label. Wherein, the common primer is used for connecting the target fragment with the adaptor and the specific label through PCR; the specific label is used for distinguishing samples, and different samples are labeled by different specific labels during library construction, so that high throughput is realized. The general sequences required by the library construction and sequencing need to be selected according to the platform library construction requirements, for example, the 5 ' end of the specific primer can be only connected with the common primer, the 3 ' end of the sequencing joint is connected with the common primer, the 3 ' end of the tag joint is connected with the common primer, and an amplicon library of ' sequencing joint-common primer-target fragment-common primer-tag joint ' can be obtained through PCR amplification; for another example, a tag and a linker are directly connected to the 5 'end of the specific primer, and the amplicon library with the specific tag and the linker can be directly obtained through PCR amplification, and in addition, only a tag is connected to the 5' end of the specific primer, and the amplicon library with the specific tag can be obtained through PCR amplification and then subjected to sequencing after linker connection; one skilled in the art will appreciate that the manner in which the specific primers are ligated 5' to the universal sequences required for library sequencing is not limited thereto.
The invention also provides a gene detection primer group for the lung cancer targeted drug, which is described by the specific primer group in the kit.
The invention provides a method for building a library of drug-targeted genes for lung cancer, which is used for non-disease diagnosis and comprises the following steps: according to the requirement of building a library of a sequencing platform, carrying out multiplex PCR amplification on template DNA by using the kit or the specific primer group to obtain an amplicon library, purifying the amplicon library, and mixing to obtain the library of the lung cancer targeted drug genes.
According to the semiconductor sequencing platform, the library building method further comprises the following steps:
(1) connecting a public primer as a specific primer group at the 5' end of a specific primer sequence shown in SEQ ID NO. 1-SEQ ID NO. 56, performing multiple PCR amplification on template DNA to obtain a first-step PCR reaction product, and purifying the first-step PCR reaction product;
(2) carrying out second-step PCR reaction on the purified first-step PCR product, a sequencing joint and a label joint to obtain a second-step PCR reaction product, purifying the second-step PCR reaction product, and mixing to obtain a library of the lung cancer targeted drug gene; wherein the tag linkers used for different template DNAs are different.
The invention also provides a library of the lung cancer targeted drug genes obtained by the library construction method.
The invention has the beneficial effects that:
the kit and the specific primer set can quickly, accurately and once detect hundreds of lung cancer targeted drug gene mutation sites, and the uniformity, the repeatability, the library building power, the library sequencing data utilization rate, the detection accuracy and the sensitivity of multiple PCR library building by using the specific primers are ideal, and the kit is suitable for various sample types.
Drawings
FIG. 1: the depth cassette map was amplified.
Detailed Description
The invention is further explained below by means of examples, without the scope of protection of the invention being limited thereto.
Reagents and equipment not specifically described in the examples are commercially available, and experimental procedures not specifically described are performed according to manufacturer's instructions or conventional techniques in the art.
Example 1 detection of specific primer set for Lung cancer Targeted drug Gene
The inventor designs specific primers according to 28 target regions of the lung cancer targeted drug related gene, and finally selects and optimizes 28 pairs of primers with high amplification efficiency and good specificity through a large number of tests, screening, optimizing and verifying (as shown in Table 1).
TABLE 1 specific primers for drug-targeted genes for lung cancer
28 target regions amplified by the specific primers are respectively used for detecting mutation sites related to the lung cancer targeted drug genes shown in table 2.
TABLE 2 mutation sites related to genes for targeted drug delivery for lung cancer
Example 2 Gene detection kit for Targeted drug administration to Lung cancer
In this embodiment, a lung cancer targeted drug gene detection kit based on a semiconductor sequencing method is exemplarily provided according to a semiconductor sequencing platform, and it should be noted that, by using the specific primer set of the present invention, development of a lung cancer targeted drug gene detection kit based on other sequencing platforms or not based on sequencing should also be a scope of protection of the present application, which is not necessarily exemplified herein.
The kit for detecting the drug-targeted gene of the lung cancer comprises:
(1) specific primer group: adding a common primer at the 5' end of each specific primer shown in SEQ ID NO. 1-SEQ ID NO. 56, wherein the common primer has the sequence: 5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 57);
(2) sequencing and connecting the joint: 5 '-CCTCTCTATGGGCAGTCGGTGAT-common primer-3' (SEQ ID NO: 58);
(3) and (3) label joint: 5 '-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT-common primer-3' (SEQ ID NO: 59); n in the sequence represents any base of AGCT, and NNNNNN is used for identifying libraries constructed by different samples;
(4) amplification buffer: commercially available Multiplex PCR buffer reagents, such as, QIAGEN Multiplex PCR Kit, 2G Fast Multiplex PCR Kit;
(5) and (3) purifying a reagent: commercially available purified magnetic beads, such as Ampure XP magnetic beads.
Example 3 library construction and sequencing method of drug targeting gene for lung cancer
The embodiment exemplarily provides a method for library construction and sequencing of a lung cancer targeted drug gene, which comprises the following steps:
1. first step PCR reaction and purification
Preparing 25 mu L of a first-step PCR reaction system, which comprises the following steps: 10-20 ng of template DNA, 2 mu L of specific primer group, 12.5 mu L of amplification buffer solution and no nuclease water in balance.
Amplification was performed according to the following procedure: firstly, the temperature is 95 ℃ for 3 min; (ii) 15 cycles of amplification, each cycle: 20s at 95 ℃, 90s at 60 ℃ and 30s at 72 ℃; keeping at 72 deg.C for 1min and at 16 deg.C.
And purifying the first-step PCR product by using a purification reagent to obtain 11.5 mu L of the purified first-step PCR product.
2. Second step PCR reaction
Preparing 25 mu L of a second-step PCR reaction system, which comprises the following steps: 11.5 mu L of purified PCR product of the first step, 0.5 mu L of sequencing joint, 0.5 mu L of label joint and 12.5 mu L of amplification buffer solution; different templates use different tag linkers.
Amplification was performed according to the following procedure: firstly, the temperature is 98 ℃ for 2 min; 25 cycles of amplification, each cycle: 10s at 98 ℃, 60s at 60 ℃ and 30s at 72 ℃; keeping at 72 deg.C for 1min and at 16 deg.C.
And (3) purifying the second-step PCR product by using a purification reagent to obtain 20 mu L of purified second-step PCR product, and mixing the second-step PCR products obtained by different templates to obtain a mixed library.
3. Sequencing
Qubit was used before sequencingTMThe dsDNA Hs Assay Kit quantifies the mixed library and performs on-machine sequencing according to the quantified concentration. In this example, a semiconductor sequencer was used for sequencing, preparation of sequencing template and enrichment detailed in Ion PITMHi QTMOT2200Kit (A26434) Instructions for use, bead Loading with template molecules to Ion ProtonTMSequencing instrumentThe semiconductor chip of (1) is subjected to sequencing, and the detailed steps are shown in Ion PITM Hi QTMThe Sequence 200Kit operation uses the instruction manual.
Example 4 index evaluation
1. Evaluation of accuracy and reproducibility
The HD734 standard (Horizon) is subjected to library construction according to the library construction method of the embodiment 3, 5 times of repetition is carried out, the obtained library is subjected to sequencing and analysis, and the result is shown in Table 3, and the 5 times of repetition can realize detection of all the variant sites, which shows that the specific primer group of the lung cancer targeted drug gene has high detection accuracy and good repeatability.
TABLE 3 test results of HD734 standard
2. Evaluation of amplification uniformity
The library is built by using the specific primer group according to the method of the embodiment 3, the obtained library is sequenced to analyze the depth of each amplicon, and the result of counting a sample in a run is shown in figure 1.
3. Evaluation of sensitivity
The sensitivity test was performed using HD780(Horizon) standard, with reference to the library building method of example 3, and the test frequency gradients were: 5%, 1%, 0.5%, 0.1%; as shown in Table 4, all known mutation sites were detected in 5%, 1% and 0.5% EGFR standards with a sensitivity of 0.5% or more, and false negative results were likely to occur when the mutation rate at a certain gene site was < 0.5%.
TABLE 4 detection results of different frequency gradients of HD780 standard substance
4. Different sample types
The DNA templates extracted for different types of samples were tested, and the data utilization and the primary library formation power were evaluated with reference to the library construction method of example 3. Wherein the sample types include: paraffin embedded tissue (FFPE), fresh tissue, pleural effusion, plasma ctDNA.
As a result, DNA samples of four specimen types were pooled at high power and the data was ideally used for the pool by sequencing analysis, as shown in Table 5.
TABLE 5 DNA test results for different sample types
| Type of sample | Number of samples | Data utilization | Primary reservoir building success rate |
| FFPE | 31 | 67.7％ | 100％ |
| Fresh tissue | 34 | 65.3％ | 100％ |
| Hydrothorax and ascites | 32 | 65.1％ | 100％ |
| Plasma ctDNA | 40 | 71.4％ | 100％ |
SEQUENCE LISTING
<110> Donggao Muohua Gene science and technology Co., Ltd
<120> gene detection kit, primers and method for lung cancer targeted medication
<130>
<160> 59
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence
<400> 1
gatggcaaat acacagagga agc 23
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
ggtgaaacct gtttgttgga cat 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
aaagtggttc tggattagct gga 23
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence
<400> 4
gctcgccaat taaccctgat tac 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence
<400> 5
ggtgttgttg tgcacaggtt att 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence
<400> 6
aaggtgaaag tctcccacaa agt 23
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence
<400> 7
agggaaaatg acaaagaaca gct 23
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence
<400> 8
tttagcactt acctgtgact cca 23
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
gcatacattc gaaagaccct agc 23
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence
<400> 10
atccattttt gttgtccagc cac 23
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence
<400> 11
cctcttacac ccagtggaga ag 22
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence
<400> 12
cagggacctt accttataca ccg 23
<210> 13
<211> 23
<212> DNA
<213> Artificial sequence
<400> 13
ggatcccaga aggtgagaaa gtt 23
<210> 14
<211> 23
<212> DNA
<213> Artificial sequence
<400> 14
acagcaaagc agaaactcac atc 23
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence
<400> 15
gcctctccct ccctccagga 20
<210> 16
<211> 22
<212> DNA
<213> Artificial sequence
<400> 16
atagtccagg aggcagccga ag 22
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence
<400> 17
caggaacgta ctggtgaaaa cac 23
<210> 18
<211> 23
<212> DNA
<213> Artificial sequence
<400> 18
cttactttgc ctccttctgc atg 23
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence
<400> 19
agcctcaatt cttaccatcc aca 23
<210> 20
<211> 23
<212> DNA
<213> Artificial sequence
<400> 20
tttcttcatg aagacctcac agt 23
<210> 21
<211> 23
<212> DNA
<213> Artificial sequence
<400> 21
ttaccatgcc actttccctt gta 23
<210> 22
<211> 23
<212> DNA
<213> Artificial sequence
<400> 22
aagaaaacac ttggtagacg gga 23
<210> 23
<211> 23
<212> DNA
<213> Artificial sequence
<400> 23
aaccatgcag atcctcagtt tgt 23
<210> 24
<211> 23
<212> DNA
<213> Artificial sequence
<400> 24
atatcaccac acacaggtaa cgg 23
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence
<400> 25
caaagaaagc cctccccagt c 21
<210> 26
<211> 25
<212> DNA
<213> Artificial sequence
<400> 26
gcaagtagta attgatggag aaacc 25
<210> 27
<211> 23
<212> DNA
<213> Artificial sequence
<400> 27
tgttggatca tattcgtcca caa 23
<210> 28
<211> 23
<212> DNA
<213> Artificial sequence
<400> 28
taaggcctgc tgaaaatgac tga 23
<210> 29
<211> 23
<212> DNA
<213> Artificial sequence
<400> 29
ccaatgaagg tgccatcatt ctt 23
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<400> 30
gagggtctga cgggtagagt 20
<210> 31
<211> 21
<212> DNA
<213> Artificial sequence
<400> 31
ataccctctc agcgtaccct t 21
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence
<400> 32
atagggcata agctgtgtca cc 22
<210> 33
<211> 19
<212> DNA
<213> Artificial sequence
<400> 33
gaaagcgcct tgaggcctt 19
<210> 34
<211> 21
<212> DNA
<213> Artificial sequence
<400> 34
cttgtgggag accttgaaca c 21
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence
<400> 35
cctgggcatc cttgagttcc 20
<210> 36
<211> 25
<212> DNA
<213> Artificial sequence
<400> 36
acttgaacca tcttttaact caggt 25
<210> 37
<211> 23
<212> DNA
<213> Artificial sequence
<400> 37
ctaggaaaga ggcaaggaaa ggt 23
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence
<400> 38
caccacgagc tgcccccagg 20
<210> 39
<211> 23
<212> DNA
<213> Artificial sequence
<400> 39
ccctttcttg cggagattct ctt 23
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence
<400> 40
ttccttactg cctcttgctt ctc 23
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence
<400> 41
ctcctgacct ggagtcttcc 20
<210> 42
<211> 23
<212> DNA
<213> Artificial sequence
<400> 42
tatctcctag gttggctctg act 23
<210> 43
<211> 22
<212> DNA
<213> Artificial sequence
<400> 43
ccactgacaa ccacccttaa cc 22
<210> 44
<211> 23
<212> DNA
<213> Artificial sequence
<400> 44
tttgcgtgtg gagtatttgg atg 23
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence
<400> 45
ctgctcacca tcgctatctg ag 22
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence
<400> 46
tgggttgatt ccacaccccc 20
<210> 47
<211> 23
<212> DNA
<213> Artificial sequence
<400> 47
caggcattga agtctcatgg aag 23
<210> 48
<211> 23
<212> DNA
<213> Artificial sequence
<400> 48
tcttctgtcc cttcccagaa aac 23
<210> 49
<211> 23
<212> DNA
<213> Artificial sequence
<400> 49
tccactcaca gtttccatag gtc 23
<210> 50
<211> 23
<212> DNA
<213> Artificial sequence
<400> 50
ctggatcccc acttttcctc ttg 23
<210> 51
<211> 23
<212> DNA
<213> Artificial sequence
<400> 51
gagggtgtct ctctgtggct tta 23
<210> 52
<211> 19
<212> DNA
<213> Artificial sequence
<400> 52
tcacagcctc ctcctcctc 19
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence
<400> 53
aaagactggt tctcactcac cg 22
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence
<400> 54
aacattgttc gctgcattgg g 21
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence
<400> 55
ggcagtcttt actcacctgt aga 23
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence
<400> 56
tgcatttcct ttcttcccag aga 23
<210> 57
<211> 18
<212> DNA
<213> Artificial sequence
<400> 57
aaatgggcgg taggcttg 18
<210> 58
<211> 23
<212> DNA
<213> Artificial sequence
<400> 58
cctctctatg ggcagtcggt gat 23
<210> 59
<211> 41
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (31)..(38)
<223> n is a, c, g, or t
<400> 59
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnga t 41
Claims (7)
1. A gene detection kit for lung cancer targeted drugs comprises: detecting a specific primer group of the lung cancer targeted drug gene; the specific primer set includes specific primers designed for target regions of genes shown in the following table:
the specific primer group comprises specific primers shown as SEQ ID NO. 1-SEQ ID NO. 56.
2. The kit of claim 1, wherein: the 5' end of each specific primer in the specific primer group is also connected with a universal sequence required by library construction and sequencing.
3. The kit of claim 2, wherein: the universal sequence is selected from a common primer, a joint and a specific label.
4. A gene detection primer group for lung cancer targeted drugs is characterized in that: a specific primer set for use in a kit according to any one of claims 1 to 3.
5. A method of library construction of drug-targeted genes for lung cancer, for non-disease diagnostic purposes, comprising the steps of: according to the requirement of a sequencing platform library, the kit of any one of claims 1 to 3 or the specific primer group of claim 4 is used for carrying out multiplex PCR amplification on the template DNA to obtain an amplicon library, and the amplicon library is purified and mixed to obtain the library of the lung cancer targeted drug genes.
6. The library building method of claim 5, wherein: the method further comprises the steps of:
(1) connecting a public primer as a specific primer group at the 5' end of a specific primer sequence shown in SEQ ID NO. 1-SEQ ID NO. 56, performing multiple PCR amplification on template DNA to obtain a first-step PCR reaction product, and purifying the first-step PCR reaction product;
(2) carrying out second-step PCR reaction on the purified first-step PCR product, a sequencing joint and a label joint to obtain a second-step PCR reaction product, purifying the second-step PCR reaction product, and mixing to obtain a library of the lung cancer targeted drug gene; wherein the tag linkers used for different template DNAs are different.
7. A library of lung cancer drug-targeting genes obtained using the library construction method of claim 5 or 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811472985.2A CN109486951B (en) | 2018-12-04 | 2018-12-04 | Lung cancer targeted drug gene detection kit, primers and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811472985.2A CN109486951B (en) | 2018-12-04 | 2018-12-04 | Lung cancer targeted drug gene detection kit, primers and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109486951A CN109486951A (en) | 2019-03-19 |
| CN109486951B true CN109486951B (en) | 2020-01-07 |
Family
ID=65699230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811472985.2A Active CN109486951B (en) | 2018-12-04 | 2018-12-04 | Lung cancer targeted drug gene detection kit, primers and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109486951B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2971093A2 (en) * | 2013-03-15 | 2016-01-20 | Life Technologies Corporation | Classification and actionability indices for lung cancer |
| CN105969857A (en) * | 2016-05-12 | 2016-09-28 | 中国科学院合肥物质科学研究院 | Non-small cell lung cancer targeted therapy gene detection method |
| CN106834286A (en) * | 2017-04-05 | 2017-06-13 | 北京泛生子基因科技有限公司 | The primer combination of one-step method rapid build amplification sublibrary |
| CN106834444A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | A kind of lung cancer related gene mutation detection methods, primer and reagent |
| CN107400714A (en) * | 2017-08-21 | 2017-11-28 | 广州永诺生物科技有限公司 | The multiple PCR primer group and kit of colorectal cancer medication related gene detection |
| CN108315416A (en) * | 2018-03-02 | 2018-07-24 | 中国科学院合肥物质科学研究院 | Primer, kit and the method for lung cancer gene mutation site are determined based on high throughput sequencing technologies |
-
2018
- 2018-12-04 CN CN201811472985.2A patent/CN109486951B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2971093A2 (en) * | 2013-03-15 | 2016-01-20 | Life Technologies Corporation | Classification and actionability indices for lung cancer |
| CN105969857A (en) * | 2016-05-12 | 2016-09-28 | 中国科学院合肥物质科学研究院 | Non-small cell lung cancer targeted therapy gene detection method |
| CN106834444A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | A kind of lung cancer related gene mutation detection methods, primer and reagent |
| CN106834286A (en) * | 2017-04-05 | 2017-06-13 | 北京泛生子基因科技有限公司 | The primer combination of one-step method rapid build amplification sublibrary |
| CN107400714A (en) * | 2017-08-21 | 2017-11-28 | 广州永诺生物科技有限公司 | The multiple PCR primer group and kit of colorectal cancer medication related gene detection |
| CN108315416A (en) * | 2018-03-02 | 2018-07-24 | 中国科学院合肥物质科学研究院 | Primer, kit and the method for lung cancer gene mutation site are determined based on high throughput sequencing technologies |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109486951A (en) | 2019-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CZ293278B6 (en) | Method for producing complex DNA methylamino fingerprints | |
| CN110004225B (en) | Tumor chemotherapeutic drug individualized gene detection kit, primers and method | |
| US20210095393A1 (en) | Method for preparing amplicon library for detecting low-frequency mutation of target gene | |
| CN102586456B (en) | Method for detecting copy number variations through multiple competitive polymerase chain reaction (PCR) | |
| CN111073961A (en) | High-throughput detection method for gene rare mutation | |
| CN105039322B (en) | DNA sequence labels and sequencing library construction method and kit | |
| WO2018147438A1 (en) | Pcr primer set for hla gene, and sequencing method using same | |
| CN113981056A (en) | Method for performing high-throughput sequencing based on internal reference of known tag | |
| CN110241215A (en) | A kind of primer, kit and detection method to make a variation for detecting Benign Thyroid Nodules tumor- associated gene | |
| CN106191311A (en) | A kind of quick detection Cavia porcellus LCMV, SV, PVM, Reo 3 virus multiple liquid phase method for gene chip and reagent | |
| CN110951851A (en) | Primer set and method for microsatellite instability detection | |
| CN108103143B (en) | Method for constructing multiple PCR and rapid library in target region | |
| CN109486951B (en) | Lung cancer targeted drug gene detection kit, primers and method | |
| CN106636442B (en) | Human tumor gene variation joint detection kit | |
| CN112266963B (en) | Detection kit for combined detection of chronic granulocytic leukemia | |
| CN112592965B (en) | E.coli host DNA residue detection kit adopting TaqMan probe method | |
| WO2019084998A1 (en) | Kit for detecting kras, nras, or braf gene mutation | |
| CN109837274B (en) | Method for constructing human mitochondrial genome library and application | |
| CN102943109A (en) | Method for detecting copy number variation based on multiple internal controls in series | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN111440876A (en) | Kit and method for quantitatively detecting methylation degree of human MGMT gene | |
| CN108330171A (en) | Detect kit, the method and its application of the 19th exon deletion mutation of EGFR gene | |
| CN108504722A (en) | A method of tandem repetitive sequence is quickly detected based on lock probe isothermal duplication | |
| CN109797210A (en) | A kind of detection method and its primer sets that Y chromosome is micro-deleted | |
| CN113502332B (en) | Primer, probe and kit for detecting FLT3 gene mutation |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |