CN109486951A - A kind of lung cancer targeting medication gene detecting kit, primer and method - Google Patents
A kind of lung cancer targeting medication gene detecting kit, primer and method Download PDFInfo
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Abstract
The invention discloses lung cancer targeting medication gene detecting kit, primer and methods.The present invention devises specific primer group for 28 target areas of lung cancer targeting medication gene, and provide lung cancer targeting medication gene detecting kit, kit and specific primer group of the invention can quickly, accurately, disposably detect several hundred a lung cancer targeting medication gene mutation sites, and multiplex PCR is carried out using specific primer and builds that the homogeneity in library, repeatability, to build Kucheng's power, library sequencing data utilization rate, detection accuracy, sensitivity ideal, and is suitable for a variety of sample types.
Description
Technical field
The invention belongs to genetic test field, relates more specifically to a kind of lung cancer targeting medication gene detecting kit, draws
Object and method.
Background technique
The main reason for lung cancer is cancer mortality, according to statistics, Chinese population lung cancer morbidity 733.3 ten thousand in 2015 are dead
610.2 ten thousand, occupy first.Genotyping can instruct lung cancer diagnosis and treatment, and genetic test instructs lung cancer therapy to have become at present
The new model of medical treatment.For example, American National comprehensive cancer network (NCCN) publication guide is pointed out, when non-small cell lung cancer diagnosis and treatment,
Need to carry out EGFR, the genetic tests such as ALK；When EGFR sensitizing mutation, EGFR-TKIs targeting medicine is recommended as a line medicine；KRAS
In the presence of mutation, it is not recommended that target medicine using EGFR-TKIs.In addition, there are also studies have shown that by genetic test guiding treatment
Patients with lung cancer Overall survival and progression free survival phase be apparently higher than the patient of traditional treatment.Therefore, exploitation is able to detect lung cancer
The product for targeting medication gene hot mutant site, can improve the life span of patients with lung cancer.
The development of high throughput target sequence capture sequencing technologies of new generation has been widely used in different kind organism research
In medical research.Compared to probe capture, using multiplex PCR capture target sequence have it is easy to operate, at low cost, the short time is big
The significant advantages such as amount enrichment target dna sequence, substantially reduce testing process and detection time.However, in general, it is multiple
The quasi- capture target area PCR is more, then specificity, accuracy, the homogeneity expanded is lower, the realization of multiplex PCR capture technique
Difficulty is bigger.
Summary of the invention
The purpose of the present invention is to provide a kind of lung cancer targeting medication gene detecting kit, primer and methods.
The technical solution used in the present invention is:
The present invention provides a kind of lung cancer to target medication gene detecting kit, comprising: detection lung cancer targets medication gene
Specific primer group；The specific primer group includes being designed for the target area of gene shown in specification table 1
Specific primer.
Preferred as mentioned reagent box, the specific primer group includes SEQ ID NO:1~SEQ ID NO:56 institute
The specific primer shown.
According to library demand is built, every specific primer 5 ' end in the specific primer group, which is also connected with, builds library sequencing institute
The universal sequence needed.The universal sequence is selected from consensus primer, connector, special signature.Wherein, the effect of consensus primer is logical
Crossing PCR connect target fragment with connector and special signature；Special signature is then used to distinguish sample, in library construction,
Different samples go to mark with different special signatures', to realize high throughput.Universal sequence needed for building library sequencing above
It needs to build demand selection in library according to platform, for example, can only connect consensus primer at 5 ' ends of specific primer, and is being sequenced
3 ' connection consensus primers of connector, 3 ' end connection consensus primers of label connector, can obtain " sequence measuring joints-public affairs by PCR amplification
The amplification sublibrary of primer-target fragment-consensus primer-label connector altogether "；For another example, directly connect at 5 ' ends of specific primer
Label and connector are connect, the amplification sublibrary with special signature and connector can be directly obtained by PCR amplification, in addition, in spy
5 ' ends of specific primer only connect label, and the amplification sublibrary for having special signature can be obtained by PCR amplification, be connected through connector
It is sequenced again after connecing；Those skilled in the art should know general sequences needed for library sequencing is built in the connection of the end of specific primer 5 '
The mode of column is without being limited thereto.
The present invention also provides a kind of lung cancer to target medication genetic test primer sets, such as the specific primer in above-mentioned kit
Described in group.
The present invention provides a kind of banking process of lung cancer targeting medication gene, and the method is used for non-disease diagnostic purpose,
Comprising steps of building library demand according to microarray dataset, using mentioned reagent box or above-mentioned specific primer group, template DNA is carried out
Multiplexed PCR amplification obtains amplification sublibrary, purifies to amplification sublibrary, and lung cancer targeting medication gene is obtained after mixing
Library.
According to semiconductor microarray dataset, above-mentioned banking process further comprises step:
(1) end of the specific primer sequence 5 ' shown in SEQ ID NO:1~SEQ ID NO:56 connection consensus primer is made
For specific primer group, multiplexed PCR amplification is carried out to template DNA, obtains first step PCR reaction product, first step PCR is reacted
Product is purified；
(2) by first step PCR product after purification, second step PCR is carried out with sequence measuring joints and label connector and is reacted, is obtained
Second step PCR reaction product purifies second step PCR reaction product, and the text of lung cancer targeting medication gene is obtained after mixing
Library；Wherein, label connector used in different templates DNA is different.
The present invention also provides the libraries of the lung cancer obtained using above-mentioned banking process targeting medication gene.
The beneficial effects of the present invention are:
The present invention devises specific primer group for 28 target areas of lung cancer targeting medication gene, and provides lung
Cancer targets medication gene detecting kit, and kit of the invention and specific primer group quickly, accurately, can be detected disposably
Several hundred a lung cancer target medication gene mutation site, and carry out multiplex PCR using specific primer and build the homogeneity in library, repetition
Property, to build Kucheng's power, library sequencing data utilization rate, detection accuracy, sensitivity ideal, and be suitable for a variety of samples
This type.
Detailed description of the invention
Fig. 1: amplification depth boxlike figure.
Specific embodiment
The present invention is explained further by the following examples, but the scope of the present invention is not limited thereto.
Not specified reagent and instrument are commercially available in embodiment, and not specified experimental implementation presses production
Quotient's specification or this field routine techniques are implemented.
The specific primer group of embodiment 1, detection lung cancer targeting medication gene
Inventor devises specific primer according to 28 target areas that lung cancer targets medication related gene, and through excessive
Experiment sieving, optimization, verifying are measured, it is final preferably to go out amplification efficiency height, 28 pairs of good primers (as shown in table 1) of specificity.
The specific primer of table 1, lung cancer targeting medication gene
28 target areas of primer amplified are respectively used to the targeting medication gene-correlation of lung cancer shown in detection table 2
Mutational site.
The mutational site of table 2, lung cancer targeting medication gene-correlation
Embodiment 2, lung cancer target medication gene detecting kit
The present embodiment illustratively provides a kind of lung cancer targeting based on semiconductor PCR sequencing PCR according to semiconductor microarray dataset
Medication gene detecting kit, it is notable that utilize specific primer group of the invention, exploitation is put down based on other sequencings
Platform or be not based on sequencing lung cancer targeting medication gene detecting kit should also be as be the application protection scope, herein not one by one
Example.
Lung cancer targets medication gene detecting kit, comprising:
(1) specific primer group: 5 ' ends of every specific primer shown in SEQ ID NO:1~SEQ ID NO:56
In addition consensus primer, the consensus primer sequence are as follows: 5 '-AAATGGGCGGTAGGCTTG-3 ' (SEQ ID NO:57)；
(2) sequence measuring joints: 5 '-CCTCTCTATGGGCAGTCGGTGAT- consensus primers -3 ' (SEQID NO:58)；
(3) label connector: 5 '-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT- consensus primers -3 '
(SEQ ID NO:59)；N indicates any base of AGCT, the library that different samples construct for identification NNNNNNNN in sequence；
(4) amplification buffer: commercially available multiplex PCR buffer reagent, e.g., QIAGEN Multiplex PCR Kit, 2G
Fast Multiplex PCR Kit；
(5) purified reagent: commercially available purifying magnetic bead, such as Ampure XP magnetic bead.
Embodiment 3, a kind of lung cancer targeting medication gene build library and sequencing approach
The present embodiment is exemplary a kind of lung cancer targeting medication gene is provided build library and sequencing approach, steps are as follows:
1, first step PCR reaction and purifying
Prepare 25 μ L first step PCR reaction systems, comprising: 10~20ng of template DNA, 2 μ L of specific primer group, amplification are slow
12.5 μ L of fliud flushing, nuclease free water surplus.
It is expanded by following programs: 1. 95 DEG C of 3min；2. 15 cyclic amplifications, each circulation: 95 DEG C of 20s, 60 DEG C
90s, 72 DEG C of 30s；3. 72 DEG C of 1min, 4. 16 DEG C of holdings.
First step PCR product is purified using purified reagent, obtains the first step PCR product of 11.5 μ L after purification.
2, second step PCR reacts
Prepare 25 μ L second step PCR reaction systems, comprising: 11.5 μ L of first step PCR product after purification, sequence measuring joints
0.5 μ L, 0.5 μ L of label connector, 12.5 μ L of amplification buffer；Different templates use different label connectors.
It is expanded by following programs: 1. 98 DEG C of 2min；2. 25 cyclic amplifications, each circulation: 98 DEG C of 10s, 60 DEG C
60s, 72 DEG C of 30s；3. 72 DEG C of 1min, 4. 16 DEG C of holdings.
Second step PCR product is purified using purified reagent, obtains the second step PCR product of 20 μ L after purification, it will
The second step PCR product that different templates obtain is mixed, and mixing library is obtained.
3, it is sequenced
Qubit is used before sequencingTMDsDNA Hs Assay Kit quantifies mixing library, according to quantitative concentration
The sequencing of the machine of progress.The present embodiment is sequenced using semiconductor sequenator, and sequencing template preparation and enrichment are detailed in Ion PITM
Hi QTMOT2200Kit (A26434) operates with specification, and the microballon with template molecule is loaded into Ion ProtonTMIt surveys
It is sequenced on the semiconductor chip of sequence instrument, detailed step is referring to Ion PITM Hi QTMSequence 200Kit is operated with
Specification.
Embodiment 4, metrics evaluation
1, accuracy and reproducibility
HD734 standard items (Horizon) are carried out building library according to the banking process of embodiment 3,5 repetitions is carried out, will obtain
The library obtained is sequenced and is analyzed, and the results are shown in Table 3, it is seen that 5 repetitions are able to achieve all variant sites detections, explanation
The specific primer group detection accuracy that lung cancer targets medication gene is high, reproducible.
Table 3, HD734 standard items testing result
2, amplification homogeneity evaluation
It carries out building library, each amplicon of library sequencing analysis of acquisition referring to the method for embodiment 3 using specific primer group
Depth, count the result of sample in a run as shown in Figure 1, amplicon depth uniformity is good, illustrate specific primer group
Primer specificity is high, interferes with each other small.
3, sensitivity evaluation
Sensitivity test is carried out using HD780 (Horizon) standard items, referring to the banking process of embodiment 3, test frequency
Gradient is respectively as follows: 5%, 1%, 0.5%, 0.1%；Testing result is as shown in table 4, and 5%, 1% and 0.5%EGFR standard items can be examined
Whole known mutations site out, sensitivity are >=0.5%, as the mutation rate < 0.5% of some gene loci, may be generated
False negative result.
Table 4, HD780 standard items different frequency gradient testing result
4, different sample types
It is tested for the DNA profiling of different types of sample extraction, referring to the banking process of embodiment 3, review number
Kucheng's power is built with primary according to utilization rate.Wherein, sample type includes: paraffin-embedded tissue (FFPE), flesh tissue, chest and abdomen
Water, blood plasma ctDNA.
The results are shown in Table 5, and it is high that the DNA sample of four kinds of sample types builds Kucheng's power, and through sequencing analysis data benefit
With library ideal.
The DNA testing result of table 5, different sample types
| Sample type | Sample number | Data user rate | Once build Kucheng's power |
| FFPE | 31 | 67.7% | 100% |
| Flesh tissue | 34 | 65.3% | 100% |
| Pleural effusions | 32 | 65.1% | 100% |
| Blood plasma ctDNA | 40 | 71.4% | 100% |
SEQUENCE LISTING
<110>Dongguan Bo Aomuhua Gene Tech. Company Limited
<120>a kind of lung cancer targeting medication gene detecting kit, primer and method
<130>
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Claims (8)
1. a kind of lung cancer targets medication gene detecting kit, comprising: the specific primer group of detection lung cancer targeting medication gene；
The specific primer group includes the specific primer being designed for the target area of gene shown in specification table 1.
2. kit according to claim 1, it is characterised in that: the specific primer group include SEQ ID NO:1~
Specific primer shown in SEQ ID NO:56.
3. kit according to claim 1 or 2, it is characterised in that: every specificity in the specific primer group
Primer 5 ', which is held to be also connected with, builds the required universal sequence of library sequencing.
4. kit according to claim 3, it is characterised in that: the universal sequence is selected from consensus primer, connector, special
Property label.
5. a kind of lung cancer targets medication genetic test primer sets, it is characterised in that: the reagent as described in any one of Claims 1 to 4
Described in specific primer group in box.
6. a kind of banking process of lung cancer targeting medication gene, the method are used for non-disease diagnostic purpose, comprising steps of according to
Microarray dataset builds library demand, is drawn using specificity described in the described in any item kits of Claims 1 to 44 or claim 5
Object group carries out multiplexed PCR amplification to template DNA, obtains amplification sublibrary, purifies to amplification sublibrary, obtains after mixing
The library of lung cancer targeting medication gene.
7. banking process according to claim 6, it is characterised in that: the method further includes steps:
(1) end of the specific primer sequence 5 ' shown in SEQ ID NO:1~SEQ ID NO:56 connection consensus primer is as special
Specific primer group carries out multiplexed PCR amplification to template DNA, first step PCR reaction product is obtained, to first step PCR reaction product
It is purified；
(2) by first step PCR product after purification, second step PCR is carried out with sequence measuring joints and label connector and is reacted, obtains second
PCR reaction product is walked, second step PCR reaction product is purified, the library of lung cancer targeting medication gene is obtained after mixing；Its
In, label connector used in different templates DNA is different.
8. utilizing the library for the lung cancer targeting medication gene that any one of claim 6 or 7 banking process obtain.
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