CN107164513A - Mankind's polygenic mutation detection kit based on high-flux sequence method - Google Patents

Mankind's polygenic mutation detection kit based on high-flux sequence method Download PDF

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CN107164513A
CN107164513A CN201710482291.6A CN201710482291A CN107164513A CN 107164513 A CN107164513 A CN 107164513A CN 201710482291 A CN201710482291 A CN 201710482291A CN 107164513 A CN107164513 A CN 107164513A
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刘蕊
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Shanghai Singlera Biotechnology Co Ltd
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Abstract

The invention belongs to biological technical field, and in particular to a kind of mankind's polygenic mutation detection kit（High-flux sequence method）And application thereof.The kit includes KRAS gene mutation detection primer to, EGFR genetic mutation detection primer pair and ALK gene fusion detection primer pair.The kit of the present invention can detect DNA mutation and RNA fusions simultaneously, and sensitivity is high, and test limit is low, and specificity is high, reproducible, and accuracy rate and positive coincidence rate are up to 100%.

Description

Mankind's polygenic mutation detection kit based on high-flux sequence method
Technical field
The invention belongs to biological technical field, and in particular to (high pass is measured a kind of mankind's polygenic mutation detection kit Sequence method) and application thereof.
Background technology
Lung cancer is to seriously endanger one of common cancer of human health, and wherein 80%-85% is non-small cell lung cancer (nonsmall cell lung cancer, NSCLC).It is continuous with what is studied Tumorigenesis and its biological behaviour Deeply, targeted therapy turns into study hotspot, multiple gene targets such as including EGFR, KRAS, ALK.
EGF-R ELISA (epidermal growth factor receptor, EGFR) is current oncotherapy Important target spot.EGFR gene is located at No. 7 the short arm of a chromosome (7p12) of people, is about 118kb, is made up of 28 extrons, its Transcribing the intracellular region of the cross-cell membrane glycoprotein of coding has EGFR-TK (tyrosine kinase, TK) activity, can pass through 2 approach transmit signals to nucleus, and one is Ras->Raf->MAPK, one is PI3K->PKC->IKK approach, and then Adjust gene transcription level in core, the propagation of influence cell, migration, differentiation, angiogenesis.The exception of EGFR signal transductions is to lead Cause the reason for kinds of tumors occurs.EGFR genetic mutation is occurred mainly on 4 extrons in intracellular TK regions (to be shown outside 18-21 Son), research shows, wherein the patients with lung cancer that 18,19,21 extrons are undergone mutation takes tyrosine kinase inhibitor The curative effect of (tyrosine kinase inhibitors, TKIs) class medicine preferably, the mutation that 20 extrons occur often with a generation, Two generation EGFR-TKIs resistances are related, but to 3 generation EGFR-TKIs class medicaments insensitives.In lung cancer patients in China, EGFR mutation rates About 30%-50%, wherein the point mutation on 18 extrons accounts for occurring on 5% or so of EGFR mutation types, 19 extrons A variety of deletion mutations T790M point mutation for accounting for occurring on 45% or so of EGFR mutation types, 20 extrons account for EGFR L858R, L861Q point mutation occurred on 5% or so of mutation type, 21 extrons accounts for the 40%- of EGFR mutation types 45%.
KRAS genes (kirsten-rous avian sarcoma) are RAS gene family members, positioned at No. 12 chromosomes 12p12.1 positions, coded product be 21KD kras albumen, participate in EGFR signal transduction processes.KRAS gene mutation can be led Cause RAS abnormal activations, induced tumor.The prompting of multiple clinical study results, the exon the 12nd of KRAS genes the 2nd, 13 passwords The mutation status of son and the curative effect that patient takes EGFR-TKIs class medicines are closely related, carry the patient of KRAS to above-mentioned The curative effect of medicine is substantially reduced.In lung cancer patients in China, KRAS gene mutation rate is about 2%-10%.
Echinoderm microtubule associated protein sample 4- anaplastic lymphoma kinases (echinoderm microtubule Associated protein like 4-anaplastic lymphoma kinase, EML4-ALK) gene fusion right and wrong Common fused type in ED-SCLC, incidence is 3%-5%, is more common in non-(slight) smoking patients with lung cancer.EML4- ALK fusion cause encode transmembrane tyrosine kinase acceptor ALK gene continuous expression so that activate alk tyrosine kinase area and The signal paths such as downstream PI3K/AKT and MAPK, cause the generation of lung cancer.Clinical research shows that ALK kinase inhibitors pass through competing Striving property is incorporated into ALK kinase regions, has blocked the signal transduction pathway in ALK downstreams so that the patient with EML4-ALK from Benefit in the treatment of ALK kinase inhibitors.
Existing lung cancer detection method of gene mutation has：
(1) Sanger sequence measurements
Concept：Sanger methods are that the point according to nucleotides in a certain fixation starts, at random at some specific base Terminate, and in each base followed by fluorescence labeling, produce with a series of cores of A, T, C, G four groups of different lengths terminated Thuja acid, then electrophoresis is detected in urea-denatured PAGE glue, so as to obtain visible DNA base sequence.
Principle is sequenced：Extended using a kind of archaeal dna polymerase with reference to the primer on sequence template undetermined.Until incorporation one Untill planting chain termination nucleotide.Sequencing is individually reacted by a set of four and constituted each time, and each reaction contains all four Deoxynucleotide triphosphoric acid (dNTP) is planted, and is mixed into a kind of different dideoxyribonucleoside triphosphate (ddNTP) of limitation.Due to DdNTP lacks the 3-OH groups required for extension, the oligonucleotide of extension is optionally terminated at G, A, T or C.Eventually Stop is in reaction depending on corresponding double deoxidation.Each dNTPs and ddNTPs relative concentration can be adjusted, and obtain reaction The chain termination product of one group long hundreds to thousands base.They have common starting point, but terminate in different nucleotides On, can be separated by high-resolution denaturing gel electrophoresis can be with x-ray film radiation certainly after fragment of different sizes, Gel Treatment Development or nonisotopic labels are detected.
Sanger method advantage and disadvantage
Advantage：Method is easy, simple to operate.
Shortcoming：The composition or secondary structure of DNA profiling cause archaeal dna polymerase abnoraml end sometimes.
(2) ARMS-PCR methods
Concept：Mutation amplification system (amplification refractory mutation system, ARMS) is also known as Allele specific amplification method (allele specific amplification, ASA), is that Newton etc. initially sets up use Method to detect known mutations.
ARMS-PCR principles：
Taq DNA polymerase lacks 3 ' -5 ' 5 prime excision enzyme activity, the mispairing of the end of PCR primer 3 ' under certain condition cause production The drastically reduction of thing, for different known mutations, differentiation mutation can be directly reached by PCR method by designing appropriate primer The purpose of type and wild type gene.
ARMS-PCR advantage and disadvantage
Advantage：Sensitivity is high, and data analysis requires low.
Shortcoming：Coverage is small, and reagent cost is high.
Genetic mechanism on lung cancer is very complicated, wherein existing DNA mutation, also there is RNA fusions.Prior art is individually detected , there is limitation in DNA mutation or RNA fusions.The combination of DNA mutation and RNA fusion detections, can increase again the economic and time into This.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide a kind of mankind's polygenic mutation Detection kit (high-flux sequence method) and application thereof.
To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：
The first aspect of the present invention is described there is provided a kind of mankind's polygenic mutation detection kit (high-flux sequence method) Kit includes DNA PCR primers and RNA PCR primers, and the DNA PCR primers include KRAS gene mutation detection primer To, EGFR genetic mutation detection primer pair, the RNA PCR primers include ALK gene fusion detection primer pair.
Preferably, the detection site of the KRAS gene mutation detection primer pair includes：c.35G>A p.G12D；c.35G> C p.G12A；c.35G>T p.G12V；c.34G>A p.G12S；c.34G>C p.G12R；c.34G>T p.G12C；c.38G>A p.G13D；The detection site of the EGFR genetic mutation detection primer pair includes：c.2573T>G p.L858R；c.2582T>A p.L861Q；c.2235_2249del p.E746_A750del；c.2236_2250del p.E746_A750del；c.2156G>C p.G719A；c.2369C>T p.T790M；The detection site of the ALK gene fusion detection primer pair include EML4 (13)- ALK(20)。
Preferably, the KRAS gene mutation detection primer to including nucleotide sequence as shown in SEQ ID NO.1 just To the reverse primer of primer and nucleotide sequence as shown in SEQ ID NO.2.The inspection of the KRAS gene mutation detection primer pair Location point includes：c.35G>A p.G12D；c.35G>C p.G12A；c.35G>T p.G12V；c.34G>A p.G12S；c.34G> C p.G12R；c.34G>T p.G12C；c.38G>Ap.G13D.
Preferably, the EGFR genetic mutation detection primer to including the first detection primer to, the second detection primer to, Three detection primers pair and the 4th detection primer pair, first detection primer is to including nucleotide sequence such as SEQ ID NO.3 institutes Reverse primer of the forward primer and nucleotide sequence shown as shown in SEQ ID NO.4, second detection primer is to including core Forward primer and nucleotide sequence reverse primer as shown in SEQ ID NO.6 of the nucleotide sequence as shown in SEQ ID NO.5, institute The 3rd detection primer is stated to forward primer and nucleotide sequence such as SEQ ID including nucleotide sequence as shown in SEQ ID NO.7 Reverse primer shown in NO.8, the 4th detection primer is drawn to the forward direction including nucleotide sequence as shown in SEQ ID NO.9 The reverse primer of thing and nucleotide sequence as shown in SEQ ID NO.10.The detection site of first detection primer pair includes c.2573T>G p.L858R and c.2582T>Ap.L861Q；C.2235_ the detection site of second detection primer pair is included 2249del p.E746_A750del、c.2236_2250del p.E746_A750del.The detection of 3rd detection primer pair C.2156G site is included>C p.G719A.C.2369C the detecting position of 4th detection primer pair is included>T p.T790M.
Preferably, the ALK gene fusion detection primer pair include nucleotide sequence as shown in SEQ ID NO.11 just To the reverse primer of primer and nucleotide sequence as shown in SEQ ID NO.12.The inspection of the ALK gene fusion detection primer pair Location point includes EML4-ALK.
The kit of the present invention can detect DNA mutation and RNA fusions simultaneously, and the design of detection primer pair is reagent of the present invention The key of box.
The present invention kit be the high throughput sequencing technologies based on ION TORRENT platforms to be detected, so Also include the conventional reagent required for some high-flux sequences based on ION TORRENT platforms in kit, such as：Library primer Mixed liquor, HIFI PCR mixed liquors, FuPa reagents, connection buffer solution, DNA ligase, HIFI enzyme mixations, elution buffer, 96) etc. (N=1,2 ... by joint, label N.
Further, the kit also includes reverse transcriptase, reverse transcriptase buffer.
Further, positive control is also contained in the kit.For example：DNA positive controls, RNA positive controls.
Further, negative control is also contained in the kit.For example：Negative control.
In a kind of embodiment of the present invention, the main constituents and its specification listed in kit are：
There is provided the application method of aforementioned agents box, including step for the second aspect of the present invention：
(1) sample nucleic acid is obtained, the nucleic acid is DNA or RNA, and reverse transcription is first carried out if nucleic acid is RNA and is cDNA；
(2) gene library is prepared：
1) multiplex PCR：The DNA that step (1) is obtained, which is used, includes the PCR reaction systems of DNA PCR primer mixed liquors Enter performing PCR amplification, obtain DNA pcr amplification products；The cDNA that step (1) is obtained is mixed using RNA PCR primers are included The PCR reaction systems of liquid enter performing PCR amplification, obtain cDNA pcr amplification products；
2) part primer sequence is digested：By step 1) the DNA pcr amplification products and cDNA pcr amplification products that are obtained, Digested using FuPa reagents, obtain postdigestive target product；
3) jointing and label：Next the target product digested by FuPa reagents is attached joint and mark Label；
4) fragment is screened：Next purifying is carried out using magnetic bead and is the DNA that nucleic acid fragment screening obtains fragment concentration, will HiFi enzyme mixations and library primer mixed liquor are well mixed, are eluted；
5) amplified library：Next the DNA for being connected to joint and label is entered into performing PCR amplification；
6) library fragments are selected：Product after PCR is expanded carries out purifying using magnetic bead and does nucleic acid fragment sieve, obtains gene Library.
The third aspect of the present invention is preparing lung cancer gene mutation and the use in fusion detection product there is provided aforementioned agents box On the way.Compared with prior art, the present invention has the advantages that：
The present invention mankind's polygenic mutation detection kit (high-flux sequence method), can detect simultaneously DNA mutation and RNA is merged, and sensitivity is high, and test limit is low, and specificity is high, reproducible, and accuracy rate and positive coincidence rate are up to 100%.
Embodiment
High-flux sequence method
High throughput sequencing technologies (High-throughput sequencing) are also known as " next generation " sequencing technologies (" Next-generation " sequencing technology), with can be once parallel to hundreds of thousands to millions of DNA molecules Carry out sequencing and general read long shorter to wait characteristic indication.
Gene mutation Gene Mutation
Unexpected, heritable variation phenomenon that i.e. genomic DNA molecule occurs.
Gene Fusion RNA Fusion
Refer to that the code area of two or more genes joins end to end, be placed in same set of regulating and controlling sequence (including promoter, enhancing Son, ribosome binding sequence, terminator etc.) control under, the mosaic gene of composition.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of unreceipted actual conditions in the following example, Generally according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment, Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The composition of the kit of embodiment 1 and preparation
Mankind's polygenic mutation detection kit (high-flux sequence method) of the present invention includes DNA PCR primer mixed liquors With RNA PCR primer mixed liquors, as shown in following table 1-1, the DNA PCR primers include KRAS gene mutation detection primer to, EGFR genetic mutation detection primer pair, the RNA PCR primers include ALK gene fusion detection primer pair, the KRAS genes Abrupt climatic change primer pair includes forward primer and the nucleotide sequence such as SEQ ID nucleotide sequence as shown in SEQ ID NO.1 Reverse primer shown in NO.2, the EGFR genetic mutation detection primer to including the first detection primer to, the second detection primer To, the 3rd detection primer pair and the 4th detection primer pair, first detection primer is to including nucleotide sequence such as SEQ ID The reverse primer of forward primer and nucleotide sequence as shown in SEQ ID NO.4 shown in NO.3, second detection primer pair It is reverse as shown in SEQ ID NO.6 including forward primer of the nucleotide sequence as shown in SEQ ID NO.5 and nucleotide sequence Primer, the 3rd detection primer is to forward primer and nucleotide sequence including nucleotide sequence as shown in SEQ ID NO.7 Reverse primer as shown in SEQ ID NO.8, the 4th detection primer to including nucleotide sequence as shown in SEQ ID NO.9 Reverse primer as shown in SEQ ID NO.10 of forward primer and nucleotide sequence, the ALK gene fusion detection primer pair It is anti-as shown in SEQ ID NO.12 including forward primer of the nucleotide sequence as shown in SEQ ID NO.11 and nucleotide sequence To primer.
The present invention kit be the high throughput sequencing technologies based on ION TORRENT platforms to be detected, so Also include the conventional reagent required for some high-flux sequences based on ION TORRENT platforms in kit, such as：Library primer Mixed liquor, HIFI PCR mixed liquors, FuPa reagents, connection buffer solution, DNA ligase, HIFI enzyme mixations, elution buffer, 96) etc. (N=1,2 ... by joint, label N.Further, the kit also includes reverse transcriptase, reverse transcriptase buffer. Further, positive control is also contained in the kit.For example：DNA positive controls, RNA positive controls.Further, institute State and also contain negative control in kit.For example：Negative control.
Can use in kit of the present invention includes DNA PCR primers and RNA PCR primers, and the DNA in sample is detected Site and RNA detection sites are expanded, and then carry out high-flux sequence to amplified production using ION TORRENT platforms.
Table 1-1
The kit of embodiment 2 and its performance evaluation test
First, the composition of kit：
The present embodiment provides a kind of specific embodiment of kit of the present invention, in the specific implementation, this The main constituents of mankind's polygenic mutation detection kit (high-flux sequence method) of invention are as shown in following table 1-2, still It is not limited thereto, as long as mankind's polygenic mutation detection kit (high-flux sequence method) includes the DNA shown in table 1-1 PCR primer and RNA PCR primers：
Table 1-2
Main constituents Specification
Library primer mixed liquor 192 μ L/ are managed, 1 pipe
HIFI PCR mixed liquors 384 μ L/ are managed, 1 pipe
FuPa reagents 192 μ L/ are managed, 1 pipe
Connect buffer solution 384 μ L/ are managed, 1 pipe
DNA ligase 192 μ L/ are managed, 1 pipe
HIFI enzyme mixations 1600 μ L/ are managed, 3 pipes
DNA PCR primer mixed liquors 192 μ L/ are managed, 1 pipe
RNA PCR primer mixed liquors 192 μ L/ are managed, 1 pipe
Elution buffer 12mL/ is managed, 1 pipe
Reverse transcriptase 48 μ L/ are managed, 1 pipe
Reverse transcriptase buffer 96 μ L/ are managed, 1 pipe
Joint 192 μ L/ are managed, 1 pipe
(N=1,2 ... 96) by label N 2 μ L/ are managed, 96 pipes
DNA positive controls 8 μ L/ are managed, 1 pipe
Negative control 16 μ L/ are managed, 1 pipe
RNA positive controls 50ng/ is managed, 2 pipes
Library purifies magnetic bead 960 μ L/ are managed, 16 pipes
Wherein, the DNA PCR primers mixed liquor in upper table 1-2 is that DNA PCR primers are dissolved in LOW TE to obtain, RNA PCR primer mixed liquor is that RNA PCR primers are dissolved in LOW TE to obtain.The DNA PCR primers mixed liquor and the RNA PCR primer mixed liquor is expanded for purpose fragment.DNA PCR primers mixed liquor and RNA PCR primers are detected using Nanodrop The concentration of mixed liquor, as a result in DNA PCR primers mixed liquor each DNA PCR primers concentration >=4000ng/ul, RNA PCR draw Concentration >=1000ng/ul of RNA PCR primers, meets use requirement in thing mixed liquor.QPCR testing results, each Ct values ＜ 35 in QPCR holes, represents to contain target primer in DNA PCR primers mixed liquor and RNA PCR primer mixed liquors.
In upper table 1-2, the library primer mixed liquor is used to carry out amplified library to the DNA for being connected to joint and label. The library primer mixed liquor can be the conventional reagent of this area, and function is with Ion Library Amplification Primer Mix。
Reverse transcriptase and reverse transcriptase buffer in upper table 1-2, are Reverse Transcription commonly used in the art, as long as It disclosure satisfy that the requirement that reverse transcription is carried out to RNA.To occur the RNA of EML4 (13)-ALK (20) fusions as template, enter Row reverse transcription, detects cDNA amplification situations, QPCR detection Reverse Transcription performances.As a result as shown in Table 2, the reverse transcriptase and Reverse transcriptase buffer meets use requirement.
Table 2
Negative control in upper table 1-2, that is, negative quality-control product, concentration >=90ng/ul of Nanodrop detections, purity： OD260/280 is between 1.6-2.0, and 1% agarose gel electrophoresis detects clip size in more than 200bp, Proton sequencing knots The kit of the present invention gene point mutation to be detected is not present in fruit, meets the requirement as negative control.
RNA positive controls in upper table 1-2, that is, RNA positive quality control products, contain the kit of the present invention gene to be detected Position of fusion, meets the requirement as RNA positive controls.
DNA positive controls in upper table 1-2, that is, DNA positive quality control products, contain the kit of the present invention gene to be detected Mutation type, meets the requirement as DNA positive controls.
In upper table 1-2, HIFI PCR mixed liquors and HIFI enzyme mixations are used to carry out conventional mould and highly difficult template Super high-fidelity amplification, HIFI PCR mixed liquors, function is obtained with Ion AmpliSeqTMHiFi Master Mix by multiplex PCR Obtain target product；HIFI enzyme mixations, function is with KAPA HiFi high-fidelity DNA polymerases.
In upper table 1-2, FuPa reagents are used to digest DNA fragmentation, and function is with FuPa Reagent.
2nd, the application method of mentioned reagent box, it may include following steps：
(1) sample nucleic acid is obtained, the nucleic acid is DNA or RNA, and reverse transcription is first carried out if nucleic acid is RNA and is cDNA；
(2) gene library is prepared：
1) multiplex PCR：The DNA that step (1) is obtained, which is used, includes the PCR reaction systems of DNA PCR primer mixed liquors Enter performing PCR amplification, obtain DNA pcr amplification products；The cDNA that step (1) is obtained is mixed using RNA PCR primers are included The PCR reaction systems of liquid enter performing PCR amplification, obtain cDNA pcr amplification products；
2) part primer sequence is digested：By step 1) the DNA pcr amplification products and cDNA pcr amplification products that are obtained, Digested using FuPa reagents, obtain postdigestive target product；
3) jointing and label：Next the target product digested by FuPa reagents is attached joint and mark Label；
4) fragment is screened：Next purifying is carried out using magnetic bead and is the DNA that nucleic acid fragment screening obtains fragment concentration, will HiFi enzyme mixations and library primer mixed liquor are well mixed, are eluted；
5) amplified library：Next the DNA for being connected to joint and label is entered into performing PCR amplification；
6) library fragments are selected：Product after PCR is expanded carries out purifying using magnetic bead and does nucleic acid fragment sieve, obtains gene Library.
Then library detection can be carried out：The concentration of amplified production is detected with Q-PCR, when library concentration is higher than machine on Proton During concentration (6pM), you can for Ion ProtonTMMicroarray dataset.
3rd, the performance evaluation test of kit：
1st, minimum detection limit scheme
Its frequency of mutation, is diluted near test limit, prepares base by 13 parts of DNA positive reference product samples with wild type sample Because of library, the upper machine sequencings of Proton；1 part of RNA positive reference product sample, test limit is diluted to wild type sample by its CP100K Near, prepare gene library, the upper machine sequencings of Proton.Criterion：Mutation or fusion higher than test limit should be detected all.
Minimum detectability (being 0.05) result of DNA PCR primers is as shown in table 4：
Table 4
Minimum detectability (being 25) result of RNA PCR primers is as shown in table 5：
Table 5
Gene Site CP100K Testing result
ALK EML4(13)-ALK(20) 162 It is positive
2nd, repeated evaluation scheme
2.1DNA PCR primers are detected：Choose 2 kinds of mutation types, i.e. EGFR (c.2236-2250del 15p.E746_ A750 del ELREA) and EGFR is (c.2573T>G p.L858R), nuclease-free water is diluted to same concentrations, mixed etc. quality Close reference and build storehouse kit into product examine.Mixed sample, continuous 10 days of 2 experiment operators repeat to build storehouse 10 times respectively, Machine is sequenced on 2 Proton respectively.
Criterion：Each mutation should be detected in sequencing result.
The repeatability of DNA PCR primers：
Table 6
2.2RNA PCR primers are detected：Choose mutation type EML4 (13)-ALK (20), 2 experiment operators continuous 10 My god, repeat to build storehouse 10 times respectively, machine is sequenced on 2 Proton respectively.
Criterion：Each mutation should be detected in sequencing result.
RNA PCR primers repeatability：
Table 7
3rd, negative and positive quality-control product：DNA and RNA negative and positive quality-control product respectively detects once that machine is sequenced on a Proton
Criterion：Sequencing result is consistent with original result.
As a result：Sequencing result is consistent with original result.
4th, it is specific：10 parts of DNA negative samples and 10 parts of RNA negative samples, build storehouse once, once respectively according to SOP The upper machine sequencings of Proton.
Criterion：It is required that sequencing result is feminine gender.
As a result：Sequencing result is feminine gender.
5th, positive coincidence rate：13 parts of DNA positive samples and 1 part of RNA positive sample, build storehouse once, once respectively according to SOP The upper machine sequencings of Proton.
Criterion：It is required that sequencing result is the positive.
DNA PCR primers positive coincidence rate is 100%：
Table 8
RNA PCR primers positive coincidence rate is 100%：
Table 9
In summary, mankind's polygenic mutation detection kit (high-flux sequence method) sensitivity of the invention is high, specifically Property it is high, reproducible, accuracy rate and positive coincidence rate are up to 100%.
It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, It should be pointed out that for those skilled in the art, on the premise of the inventive method is not departed from, can also make Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile, all substantial technologicals pair according to the present invention The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme It is interior.
SEQUENCE LISTING
<110>Shang remote Bioisystech Co., Ltd of Hai Kun
<120>Mankind's polygenic mutation detection kit（High-flux sequence method）
<130> 172305
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213> Artificial
<220>
<223>KRAS gene mutation detects forward primer
<400> 1
tacctctatt gttggatcat attcgtcca 29
<210> 2
<211> 30
<212> DNA
<213> Artificial
<220>
<223>KRAS gene mutation detects reverse primer
<400> 2
tattataagg cctgctgaaa atgactgaat 30
<210> 3
<211> 22
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the first detection primer to forward primer
<400> 3
gccaggaacg tactggtgaa aa 22
<210> 4
<211> 24
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the first detection primer to reverse primer
<400> 4
tgacctaaag ccacctcctt actt 24
<210> 5
<211> 26
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the second detection primer to forward primer
<400> 5
taacgtcttc cttctctctc tgtcat 26
<210> 6
<211> 25
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the second detection primer to reverse primer
<400> 6
agcagaaact cacatcgagg atttc 25
<210> 7
<211> 22
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the 3rd detection primer to forward primer
<400> 7
tcttacaccc agtggagaag ct 22
<210> 8
<211> 23
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the 3rd detection primer to reverse primer
<400> 8
tgtgccaggg accttacctt ata 23
<210> 9
<211> 19
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the 3rd detection primer to forward primer
<400> 9
gaagcctacg tgatggcca 19
<210> 10
<211> 22
<212> DNA
<213> Artificial
<220>
<223>EGFR genetic mutation detects the 3rd detection primer to reverse primer
<400> 10
ttgtctttgt gttcccggac at 22
<210> 11
<211> 27
<212> DNA
<213> Artificial
<220>
<223>ALK gene fusion detection primer pair forward primer
<400> 11
cctgggaaag gacctaaagg tgtatat 27
<210> 12
<211> 22
<212> DNA
<213> Artificial
<220>
<223>ALK gene fusion detection primer pair reverse primer
<400> 12
ccgaatgagg gtgatgtttt tc 22

Claims (10)

1. a kind of mankind's polygenic mutation detection kit, it is characterised in that the kit include DNA PCR primers and RNA PCR primers, the DNA PCR primers include KRAS gene mutation detection primer to, EGFR genetic mutation detection primer pair, The RNA PCR primers include ALK gene fusion detection primer pair.
2. kit according to claim 1, it is characterised in that the detecting position of the KRAS gene mutation detection primer pair Point includes：c.35G>A p.G12D；c.35G>C p.G12A；c.35G>T p.G12V；c.34G>A p.G12S；c.34G>C p.G12R；c.34G>T p.G12C；c.38G>A p.G13D；The detection site bag of the EGFR genetic mutation detection primer pair Include：c.2573T>G p.L858R；c.2582T>A p.L861Q；c.2235_2249delp.E746_A750del；c.2236_ 2250del p.E746_A750del；c.2156G>C p.G719A；c.2369C>T p.T790M；The ALK gene fusion inspection Surveying the detection site of primer pair includes EML4 (13)-ALK (20).
3. kit according to claim 1, it is characterised in that the KRAS gene mutation detection primer is to including nucleosides Forward primer and nucleotide sequence reverse primer as shown in SEQ ID NO.2 of the acid sequence as shown in SEQ ID NO.1.
4. kit according to claim 1, it is characterised in that the EGFR genetic mutation detection primer is to including first Detection primer is to, the second detection primer to, the 3rd detection primer pair and the 4th detection primer pair, and first detection primer is to bag Include forward primer and nucleotide sequence of the nucleotide sequence as shown in SEQ ID NO.3 reversely drawing as shown in SEQ ID NO.4 Thing, second detection primer to including forward primer of the nucleotide sequence as shown in SEQ ID NO.5 and nucleotide sequence such as Reverse primer shown in SEQ ID NO.6, the 3rd detection primer to including nucleotide sequence as shown in SEQ ID NO.7 The reverse primer of forward primer and nucleotide sequence as shown in SEQ ID NO.8, the 4th detection primer is to including nucleotides Forward primer and nucleotide sequence reverse primer as shown in SEQ ID NO.10 of the sequence as shown in SEQ ID NO.9.
5. kit according to claim 1, it is characterised in that the ALK gene fusion detection primer pair includes nucleosides Forward primer and nucleotide sequence reverse primer as shown in SEQ ID NO.12 of the acid sequence as shown in SEQ ID NO.11.
6. kit according to claim 1, it is characterised in that the kit includes each group primer of independent packaging It is right, or the DNA PCR primers mixed liquor containing each group primer pair, RNA PCR primer mixed liquors including having configured.
7. kit according to claim 1, it is characterised in that also include library primer mixed liquor, HIFI in kit PCR mixed liquors, FuPa reagents, connection buffer solution, DNA ligase, HIFI enzyme mixations, elution buffer, reverse transcriptase, reverse Record enzyme buffer liquid, joint, label N (N=1,2 ... 96), library purifying magnetic bead, DNA positive controls, RNA positive controls, negative right According to.
8. kit according to claim 1, it is characterised in that main constituents and its specification in kit are：
9. the application method of kit as described in claim any one of 1-8, including step：
(1) sample nucleic acid is obtained, the nucleic acid is DNA or RNA, and it is cDNA that reverse transcription is first carried out if nucleic acid is RNA；
(2) gene library is prepared：
1) multiplex PCR：The DNA that step (1) is obtained uses the PCR reaction systems for including DNA PCR primer mixed liquors to carry out PCR is expanded, and obtains DNA pcr amplification products；The cDNA that step (1) is obtained, which is used, includes RNA PCR primer mixed liquors PCR reaction systems enter performing PCR amplification, obtain cDNA pcr amplification products；
2) part primer sequence is digested：By step 1) the DNA pcr amplification products and cDNA pcr amplification products that are obtained, use FuPa reagents digest, and obtain postdigestive target product；
3) jointing and label：Next the target product digested by FuPa reagents is attached joint and label；
4) fragment is screened：Next purifying is carried out using magnetic bead and is the DNA that nucleic acid fragment screening obtains fragment concentration, by HiFi enzymes Mixed liquor and library primer mixed liquor are well mixed, are eluted；
5) amplified library：Next the DNA for being connected to joint and label is entered into performing PCR amplification；
6) library fragments are selected：Product after PCR is expanded carries out purifying using magnetic bead and does nucleic acid fragment screening, obtains gene text Storehouse.
10. kit is preparing lung cancer gene mutation and the purposes in fusion detection product as described in claim any one of 1-9.
CN201710482291.6A 2017-06-22 2017-06-22 Mankind's polygenic mutation detection kit based on high-flux sequence method Pending CN107164513A (en)

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CN109517884A (en) * 2018-09-30 2019-03-26 北京安智因生物技术有限公司 A kind of construction method and its kit in the genetic test library of familial hypercholesterolemia
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CN112080566A (en) * 2020-08-25 2020-12-15 上海交通大学医学院附属瑞金医院 Thyroid cancer detection product based on high-throughput sequencing method and application
CN114842911A (en) * 2022-06-21 2022-08-02 深圳市睿法生物科技有限公司 Gene detection process optimization method and device based on precise medical treatment
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Application publication date: 20170915