CN105256021A

CN105256021A - Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing

Info

Publication number: CN105256021A
Application number: CN201510666246.7A
Authority: CN
Inventors: 黄志清; 施纯玫; 郑建伟; 陈强; 翁芳霞; 王劲
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2015-10-16
Filing date: 2015-10-16
Publication date: 2016-01-20
Anticipated expiration: 2035-10-16
Also published as: CN105256021B

Abstract

The invention belongs to the field of gene mutation detection and particularly relates to a method and a kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on the basis of Sanger sequencing. In order to overcome the defects of a conventional human EGFR gene mutation detection method and kit, the method and the kit for flexibly detecting the human EGFR gene mutation on the basis of the Sanger sequencing are provided; heatproof DNA ligase and a specific connecting primer system aiming at a mutation site are introduced into an EGFR gene segment amplification system, so that amplification of wild type EGFR gene segments in a sample is greatly inhibited, and a wild type sample can be directly determined on the basis of amplified segment bands; or the proportion of mutation segments in a final PCR (polymerase chain reaction) product of a sample with low mutation content is enabled to be higher than 50%, accordingly, the accurate identification of the Sanger sequencing on a heterozygous gene segment system is guaranteed; the method and the kit can detect the EGFR gene mutation with the content as low as 1% in the sample on the basis of the Sanger sequencing, the accuracy rate is very high, the detection cost is low, and new unknown mutation can be possibly detected.

Description

Based on method and the test kit thereof of Sanger order-checking Sensitive Detection human EGFR gene mutations

Technical field

The invention belongs to detection in Gene Mutation field, particularly relate to the method and test kit that utilize Sanger order-checking Sensitive Detection human EGFR gene mutations.

Background technology

Human epidermal growth factor acceptor (EpidermalGrowthFactorReceptor, EGFR) be a kind of transmembrane glycoprotein with tyrosine kinase activity of being encoded by proto-oncogene C-erbB-1 (HER-1), wide expression is in most of normal epithelium cell.But in many noumenal tumours (as nonsmall-cell lung cancer), often there is the situation of EGFR high expression level or unconventionality expression, the suppression of this propagation with tumour cell, vasculogenesis, invasion and attack, transfer and apoptosis is relevant, and therefore EGFR has become an important target spot of related neoplasms (especially nonsmall-cell lung cancer) targeted therapy.Be applied to clinical EGFR targeted drug at present to comprise: EGFR tyrosine kinase inhibitor (TKIs, as Gefitinib and Iressa and Erlotinib and Erlotinib), and EGFR monoclonal antibody is as Cetuximab.Large quantity research shows, the sudden change situation of the curative effect of Gefitinib (Gefitinib) and Erlotinib (Erlotinib) and EGFR gene especially its 18-21 exon is closely related.The patients with lung cancer carrying EGFR genetic mutation is extremely sensitive to tyrosine kinase inhibitor, and patient will obtain very large being benefited; Otherwise the patient of wild type gene will not be benefited substantially, therefore EGFR gene detects and has been put into the up-to-date comprehensive cancer network of NCCN(American National as cancer target such as nonsmall-cell lung cancers to the prerequisite of choice of drug) clinical tumor practice guideline.

The EGFR genetic mutation detection method of current report comprises Sanger order-checking, Scorpions-ARMS, TaqMan-qPCR, DHPLC, PCR-SSCP/RFLP etc., wherein comparatively conventional in clinical and scientific research method is Sanger sequencing and Scorpions-ARMS (Scorpions, scorpion shape probe; Amplificationrefractorymutationsystem, amplification refractory mutation system) method.Based on the EGFR detection kit as German Qiagen company of Scorpions-ARMS method exploitation, 29 kinds of common mutations of EGFR gene can be detected simultaneously, have highly sensitive (sudden change being low to moderate 1% level can be measured), simple to operate, the advantages such as rapid detection; But such import reagent box testing cost too high (every part of sample needs 3000-5000 unit) limits its clinical promotion and application.Sanger sequencing is the gold standard that transgenation/diversity detects, and the advantage such as possess skills and platform is ripe, result is accurate and comprehensive, and compared to Scorpions-ARMS method, testing cost is cheap and can also measure unknown mutation; But the main deficiency of Sanger sequencing is that detection sensitivity is about about 10-20% (i.e. mutator gene content be greater than 10% even 20% could reliably be detected), this restrict its clinical application.Because somatic mutation component only accounts for the part in clinical equal samples, in addition also may there is a large amount of wild type genes, therefore improve the susceptibility of a small amount of abrupt climatic change in a large amount of normal gene background, be the key of improvement or development approach at present.

Summary of the invention

In order to overcome the deficiency of existing human EGFR gene mutations detection method and test kit, an object of the present invention is the test kit providing a kind of Sensitive Detection human EGFR gene mutations that checks order based on Sanger; Another object of the present invention is a kind of method providing Sensitive Detection human EGFR gene mutations based on Sanger order-checking.

Because the present invention adds heat-resistant dna ligase enzyme and the connection primer for mutational site in the amplification system of the specific fragment of EGFR gene, cause the amplification of the Wild type EGFR gene fragment in sample will be subject to effective suppression, the amplification of saltant type is then substantially unaffected; Therefore make sudden change content be low to moderate the ratio of mutant fragments in its final amplified production of sample of 1% higher than 50%, thus ensure that the accurate identification of Sanger order-checking for heterozygous genes fragment system.

The technical solution adopted for the present invention to solve the technical problems is as follows:

First the present invention relates to a kind of primer of the Sensitive Detection human EGFR gene mutations of checking order based on Sanger, described primer by amplimer group be connected primer sets and form, the nucleotide sequence of described connection primer sets is as shown in SEQ.IDNO.9-SEQ.IDNO.26.

Secondly, the test kit of the Sensitive Detection human EGFR gene mutations that checks order based on Sanger of the present invention adds described connection primer sets, heat-resistant dna ligase enzyme and damping fluid thereof in EGFR gene fragment amplification system.

More specifically, to check order the method for Sensitive Detection human EGFR gene mutations and test kit thereof based on Sanger, concrete steps and test kit associated viscera of the present invention as follows:

(1) sample collection and extracting genome DNA

Gather or take out biological specimen to be measured as the tumor tissues of patient or peripheral blood etc., utilizing corresponding test kit to extract its genomic dna.

(2) the specific fragment amplification of EGFR gene

Adopt test kit of the present invention prepare respectively the combination of EGFR gene exon containing ligase enzyme amplification system, need altogether preparation 5 to manage; All the other are all identical, as shown in table 1 except being connected primer difference except amplimer for the 5 pipe amplification systems prepared:

Table 1EGFR gene extron amplification system (25uL)

Corresponding not the managing containing the contrast amplification system 5 connecting primer, heat-resistant dna ligase enzyme damping fluid and these three kinds of components of heat-resistant dna ligase enzyme of preparation simultaneously, system preparation is with reference to table 1, scarce component addition is substituted by sterilizing deionized water, and all the other components are with corresponding normal system.

The totally 5 normal system of pipes (containing ligase enzyme system) and 5 pipe control systems (not containing ligase enzyme system) carry out the amplification of EGFR gene fragment on gene-amplificative instrament, and amplification condition is: 95 DEG C of denaturation 5-10min; 94 DEG C of sex change 15-30s, 50-53 DEG C of annealing 45-90s, 72 DEG C extend 1-2min, circulation 30-36 time; 72 DEG C of ends extend 10-12min.

(3) rubber tapping of goal gene fragment is reclaimed

All PCR primer carry out agarose gel electrophoresis, and dye 20min be placed on clear water decolouring and gel imaging system take pictures and analyzes in the EB solution of 0.5ug/ml.When control systems Successful amplification goes out goal gene fragment, with DNAMarker band for standard, select the band that in normal system, each exon amplification production concentration is not less than 10ng/uL to tap rubber, and reclaim with gel purification kit.

(4) goal gene fragment sequence measures

Product is reclaimed for rubber tapping, carries out two-way order-checking and analysis, according to the sequence of all order-checking peak figure final analysis goal gene fragments according to the standard operation of DNA fragmentation order-checking.

(5) EGFR sudden change result judges

When control systems normally increases, if each exon amplification band in normal system is all without the object band meeting rubber tapping and reclaim, then directly judge that the EGFR gene detected result of sample is as feminine gender and wild-type without the need to carry out tapping rubber recovery and subsequent operations; If there is the object band meeting rubber tapping and reclaim, and two-way sequencing result is shown as the corresponding known mutations connecting primer target, then judge that the EGFR gene detected result of sample is as the positive and saltant type, mutation type is as the criterion with the actual known mutations measured; If two-way sequencing result is same unknown difference site, then need to judge that the EGFR gene detected result of sample is as new mutant or polymorphism further, sudden change/polymorphism type with actual measure be as the criterion; If two-way sequencing result is wild-type, then judge that the EGFR gene detected result of sample is as judging, needs to redeterminate; If two-way sequencing result is one to for wild-type one is to being saltant type, then judge that the EGFR gene detected result of sample is as judging, needs to redeterminate.

Wherein, step (1) is if the acquisition target tumour patient of middle sample then should carry out under the prerequisite of its abundant informed consent; The tissue samples gathered or take out fixes paraffin-embedded tissue section including but not limited to flesh tissue, frozen tissue, formaldehyde immersion tissue and formaldehyde, blood sample is including but not limited to fresh blood, blood plasma and serum, other biological specimen is including but not limited to hydrothorax, ascites and exfoliated tumor cells, and the sampling requirement such as sample collection or the amount of taking and pre-treatment are extracted test kit according to the genome of correspondence and performed; After extracting sample genomic dna, utilize agarose gel electrophoresis or inhale brightness method and assess its extracted amount (concentration) and purity, need the effective amplification ensureing EGFR gene fragment: the extracted amount as genomic dna is no less than 200ng, and purity is with OD ₂₆₀/ OD ₂₈₀be advisable between 1.8-2.0.

Wherein, the amplification kit component adopted in step (2) is as shown in table 2:

Table 2 amplification kit component of the present invention

In table 2 component, the enzyme activity unit concentration of archaeal dna polymerase is 5U/uL, has resistance toheat; 10 × PCR damping fluid is by 100mmol/LTris-HCl(pH=8.3), 500mmol/LKCl and 15mmol/LMgCl ₂composition; DNTPs is mixed by each 2.5mmol/L of dATP, dTTP, dCTP and dGTP; Corresponding to the 18th, 19,20 and 21 exons of EGFR gene, each exon has pair for amplification primer (forward primer and reverse primer) separately, and the concentration of every bar amplimer is 10umol/L; Corresponding to the 18th, 19,20 and 21 exons of EGFR gene, each exon has the connection primer of one group of target target site (namely belonging to the site that 29 kinds of common mutations can occur) on it separately, and the concentration that every bar connects primer is 10umol/L; The enzyme activity unit concentration of heat-resistant dna ligase enzyme is not less than 4000U/uL, and under normal system, 95 DEG C of insulation 1h enzyme activities are not less than 60% of protoenzyme work; 10 × heat-resistant dna ligase enzyme damping fluid is 200mMTris-HCl, 250mM potassium acetate, 100mM magnesium acetate, 10mMNAD(Reduced nicotinamide-adenine dinucleotide), 100mMDTT(dithiothreitol (DTT)), 1%TritonX-100(Triton X-100), pH=7.6.

Wherein, the combination of step (2) Exon amplimer be connected primer and relevant information is as shown in table 3:

The amplimer of table 3EGFR exon and amplified fragments size and be connected primer and for mutation type information

As shown in table 3, adopt combination multiplex amplification under the prerequisite of guarantee 29 kinds of abrupt climatic change, can at utmost reduce required detector tube number, thus simplify the operation with cost-saving.Corresponding to table 3, amplimer is as shown in table 4 with the sequence and target information being connected primer:

Table 4 amplimer be connected primer sequence and target information

SEQ.IDNO.9 in table 4,11,13,15,17,19,21,5 ' first Nucleotide held of 23 and 25 all carries out phosphorylation modification, SEQ.IDNO.10,12,14,16,18,20,22, the 5 ' end of 24 and 26 and 3 ' first Nucleotide held all carries out phosphorylation modification.

Wherein, in step (2), provide the component for the system of 25uL and content, can as the preparation foundation of other system as systems such as 50uL.

Wherein, in step (3), the sample applied sample amount of agarose gel electrophoresis is no less than 15ul, its electrophoretic band brightness of the object fragment that control systems amplifies detects by an unaided eye, and the standard band brightness (being generally 5-10ng/uL) that should be not less than DNAMarker then represents that expanding effect is good; In addition by the concentration of each standard band of DNAMarker, if a certain fragment is 10ng/uL, then when same applied sample amount, only select the brightness object band suitable with this standard band to carry out tapping rubber and reclaim (concentration is not carried out rubber tapping lower than the object band of 10ng/uL and reclaimed), the software that can carry by gel imaging system as needs or the Quantity-one isogel analysis software of BioRad company of the U.S. carry out band Luminance Analysis.

Wherein, in step (4), the order-checking of DNA fragmentation only need be carried out according to the standard operation of Sanger order-checking, there is no and improves further or variation; Therefore can be according to actual needs, send rubber tapping to reclaim product as requested to measure to the feeler mechanism or company with qualification (as clinical gene analyzes qualification), it should be noted that and need order-checking unit to provide the original datagram of order-checking to draw together peak figure, to analyze further according to self-demand and to understand.

Wherein, the middle EGFR gene as the wild-type judged of suddenling change of step (5) and the 18th, 19,20 and 21 exon sequence canonical sequence AY588246(GeneBank accession number thereof); Corresponding to sequence A Y588246, the start-stop region of the 18th, 19,20 and 21 exon sequences is respectively 156750-156872,157551-157649,164123-164308 and 174552-174707.

Wherein, directly can be judged to be the sample of wild-type in step (5), the goal gene fragment that still normal system can be increased carries out rubber tapping order-checking, confirms further.

Wherein, in step (5), two-way sequencing result is same unknown difference site and is assumed to be adenine nucleotide A, if needed then desirable normal cell if hemocyte (except blood cancer patient) is as benchmark, conventionally measure the EGFR gene fragment sequence of its correspondence: if the result that normal cell is measured is also A, is judged to be polymorphism, if measure the result of non-A, is judged to be unknown mutation.

Wherein, step (5) if in need again to detect EGFR genetic mutation situation (namely two-way sequencing result is wild-type or to for wild-type one is to being saltant type), if the detection of employing the inventive method still cannot judge catastrophe again, then can advise using other measuring method instead.

Wherein, according to after the sample EGFR gene detected result obtained in step (5), contrast existing clinical findings or scientific research achievement, clinician informs that corresponding tumour patient predicts that it accepts the benefited situation of targeted drug treatment.

Wherein, tumour patient comprises nonsmall-cell lung cancer but is not limited to this in step (5), and targeted drug comprises Gefitinib and Erlotinib but is not limited to this.For nonsmall-cell lung cancer, the benefited situation that each single sudden change of EGFR and targeted drug Gefitinib and Erlotinib use is: 20 exons T790M sudden change occur and insert, by the resistance of prediction TKI medicine or invalid; All the other sudden changes all predict that TKI pharmacological agent is effective.

the invention has the beneficial effects as followsbased on method and the test kit thereof of Sanger order-checking Sensitive Detection human EGFR gene mutations, owing to introducing heat-resistant dna ligase enzyme and the specific connection primer system for mutational site in EGFR gene fragment amplification system, greatly inhibit the amplification of the Wild type EGFR gene in sample, can directly judge wild-type sample based on amplified fragments band; Or make the ratio of mutant fragments in low its final PCR primer of sudden change content sample higher than 50%, thus ensure that the accurate identification of Sanger order-checking for heterozygous genes fragment system; Based on Sanger order-checking, the inventive method and test kit can detect that in sample, content is low to moderate the EGFR genetic mutation of 1%, accuracy rate is high, and testing cost is cheap and may measure new unknown mutation.

Accompanying drawing explanation

Fig. 1 the present invention is based on the principle schematic connecting suppressing method.In figure, " open country " represents wild type gene group template, " dash forward " and represent mutated genes group template (the rice word symbology mutational site on it), arrow represents amplimer and bearing of trend thereof, L with R representative is connected primer (the solid initial point on it represents phosphorylation modification), trilateral representation DNA polysaccharase, circle representation DNA ligase enzyme, the representative of the tilde before 53 DEG C of annealing approximates, annealing stage L connect primer right-hand member tilt represent it and mutational site unpaired, extend the dotted arrow representation DNA chain extension that stage amplification primer extension goes out, the inclination dotted arrow representative that the extension stage connects the primer upper right corner connects primer disengaging mutated genes group template, long primer connection primer L and R that connect is formed by connecting through DNA ligase effect.

Fig. 2 is the electrophoretogram of the G719A sudden change heterozygote sample amplified production of EGFR18.M representation DNA Marker, and then the swimming lane 1-5 of Marker is respectively the amplified production of the 1-5 pipe of control systems; Swimming lane 6-10 is respectively the amplified production of the 1-5 pipe of normal system.

Fig. 3 is G719A (6239) the heterozygote order-checking peak figure local of EGFR18, and in figure, circle and arrow logo place there occurs the conversion of GC as seen.

Fig. 4 is the L747_S752del(6255 of EGFR19) heterozygote order-checking peak figure local, in figure, circle and arrow logo place there occurs disappearance as seen.

Fig. 5 be EGFR20 T790M (6240) heterozygote order-checking peak figure local, in figure, circle and arrow logo place there occurs GA(or CT as seen) transversion.

Embodiment

one, based on the method for Sanger order-checking Sensitive Detection human EGFR gene mutations and the principle of test kit thereof

Sanger sequencing is the gold standard of gene diversity/abrupt climatic change, and goal gene fragment is obtained by the primer pair amplifies of its two ends conservative region of target; For the sample of nucleic acid that composite shuttering is originated as heterozygote, if amplified fragments content wherein has be less than 10%, be difficult to accurately identify this fragment by checking order.Tumour is somatocyte disease, because somatic mutation component only accounts for the part in clinical equal samples, in addition also may there is a large amount of wild type genes, therefore adopt conventional Sanger order-checking cannot ensure the sensitivity requirement of this type of sample detection in Gene Mutation.

For the deficiency of Sanger sequencing in EGFR genetic mutation detects, the present invention proposes the method connecting suppression and improve, Fig. 1 is its principle schematic.The present invention with 29 of EGFR gene kinds of common mutations for object, devise the connection primer (SEQ.IDNO.9-SEQ.IDNO.18) in each mutational site of target, make each to connect the corresponding mutational site of primer and wild type gene region adjacent complementation (two be connected primer all with template complete complementary and adjoin).Owing to introducing heat-resisting DNA ligase in the amplification system of EGFR gene exon, then time annealing stage (~ 50 DEG C), DNA ligase will connect the complementary primer that adjoins be positioned on wild-type template and forms long connection primer, due to two that are combined with saltant type template connect primer adjacent place (i.e. mutational site place) not and template complete complementary be not then connected.When system be in next stage that is 72 DEG C extend time, owing to being incorporated into the long primer annealing temperature that connects on wild-type template higher than 72 DEG C, being therefore still positioned at original place thus stoping the amplification of wild-type template; And for saltant type template, be two short connection primers due to what combine, its annealing temperature only has about 60 DEG C, and at the temperature of 72 DEG C, they will be separated with template, thus can not hinder the amplification of template.

Assuming that the content of wild-type template has 100 in tumor sample genome, mutagenesis template content only has 1, and object fragment increases with the index power of 2; If the average suppression efficiency increased for wild-type template to connect primer is only 20%, then the amplification output of wild-type object fragment is 100 × [2 × (1-20%)] ⁿ(n is amplification cycles number), the amplification output of saltant type object fragment is 2 ⁿ; Through 21 circulations, the content of saltant type object fragment will exceed wild-type object fragment.It can thus be appreciated that the amplification of the Wild type EGFR gene fragment made in final sample is subject to effective suppression by the inventive method, the amplification of saltant type is then substantially unaffected; Therefore make sudden change content be low to moderate the ratio of mutant fragments in its final amplified production of sample of 1% higher than 50%, thus ensure that the accurate identification of Sanger order-checking for heterozygous genes fragment system.

The present invention constructs Circulating DNA polymerization first and is connected associating system with Circulating DNA, and optimize content and the ratio of polymerization and each component of linked system further, the connection primer of the EGFR genetic mutation that the target that final combination designs is common is introduced in the detection of EGFR genetic mutation; Wherein each technical parameter and application constitute the test kit corresponding to the inventive method.

two, embodiment

Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited to this.

The present embodiment increases the EGFR gene fragment that obtains for wild-type template with Healthy People hemocyte, is cloned in colibacillus engineering; Overlapping extension PCR is utilized to obtain the EGFR gene fragment of saltant type, be cloned in colibacillus engineering, construct EGFR gene wild-type and mutant cell system thus respectively, set up the method based on Sanger order-checking Sensitive Detection human EGFR gene mutations and test kit thereof on this basis.Mainly comprise the following steps:

(1) the selecting of each component in test kit.

The each component of test kit of the present invention selects information as shown in table 5.

Table 5 reagent constituents selects information

In table 5 component, the enzyme activity unit concentration of rTaqDNA polysaccharase is the enzyme activity unit concentration of 5U/uL, TaqDNA ligase enzyme is 4000U/uL, and both all have good resistance toheat.

(2) preparation of heterozygosis sample and extracting genome DNA

Reference literature (Xiao Li, EGFR gene deletion mutantion new detecting technique and be applied to dissociative DNA detect feasibility. University Of Suzhou Ph.D. Dissertation, 2014; Zhao Jing, the exploitation of nonsmall-cell lung cancer EGFR genetic mutation detection kit. Beijing Union Medical College Ph.D. Dissertation, 2011) in method build containing common 29 kinds of saltant types in EGFR gene 18-21 tetra-exon wild-types and its e.colidH5 α engineering bacteria clone, obtains by TA clone, the screening of blue hickie and order-checking qualification the positive proceeding to object fragment e.colidH5 α clone.Then positive colony is cultivated with LB liquid nutrient medium, total wild-type bacteria (being called for short wild-type bacteria below) is mixed to get, the wild-type bacteria in total: saltant type bacteria concentration mixes than for 100:1 the heterozygote sample namely obtained containing 1% mutant cell ratio according to EGFR gene 18-21 tetra-exon wild-type bacteria equal proportions.That the present embodiment is selected is EGFR18 i.e. G719A (6239), the EGFR19 i.e. L747_S752del(6255 of 19 exons of 18 exons) and the mixed bacterium of the EGFR20 i.e. T790M (6240) of 20 exons totally 3 kinds of mutation types, for EGFR21 i.e. 21 exons then only use wild-type bacteria; Then the present embodiment contains 3 kinds of heterozygote samples and a kind of homozygote sample totally 4 samples.Adopt the MiniBESTBacteriaGenomicDNAExtractionKitVer.3.0 of TaKaRa company to extract the genomic dna of sample, obtain 50uL altogether.The electrophoresis of each sample genomic dna does not occur degraded and brightness is fine, proves that extraction effect is good; After measured extracted amount be 1-2ug, A260/A280 all between 1.8-2.0, meet the amount needed for amplification and purity.

(3) the specific fragment amplification of EGFR gene

Adopt test kit of the present invention to prepare 5 above-mentioned pipe amplification systems respectively, the amplimer of each body system be connected primer sets with reference to table 3, all the other components are all identical as shown in table 6:

Table 6EGFR gene extron amplification system (25uL)

Prepare 5 corresponding pipes not containing the contrast amplification system connecting these three kinds of components (ligase enzyme system) of primer, heat-resistant dna ligase enzyme damping fluid and heat-resistant dna ligase enzyme simultaneously, amplimer is with reference to table 3, system preparation is with reference to table 6, and scarce component addition is substituted by sterilizing deionized water.

For each sample, the totally 5 normal system of pipes (containing ligase enzyme system) and 5 pipe control systems (not containing ligase enzyme system) carry out the amplification of EGFR gene fragment on gene-amplificative instrament, and amplification condition is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 20s, 50 DEG C of annealing 60s, 72 DEG C extend 1min, circulate 32 times; 72 DEG C of ends extend 10min.

(4) rubber tapping of goal gene fragment is reclaimed

All PCR primer carry out agarose gel electrophoresis, dyeing 20min in the EB solution (0.5ug/ml) is also placed on clear water decolouring and gel imaging system takes pictures and analyzes, for the electrophoretogram of its amplified production of the G719A of EGFR18 sudden change heterozygote sample as shown in Figure 2, swimming lane 1-5 is respectively the amplified production of the 1-5 pipe of control systems; Swimming lane 6-10 is respectively the amplified production of the 1-5 pipe of normal system.With the D2000DNAMarker band of Beijing Tian Gen company for standard (common band concentration is about 10ng/uL), the control systems of the G719A sudden change of EGFR18 all can go out goal gene fragment by Successful amplification, and in normal system, only having the amplified production band bright (concentration is higher than 10ng/uL) of the 1st pipe, the amplified production band of all the other 4 pipes is faint (concentration is starkly lower than 10ng/uL).For all the other two kinds sudden change heterozygote templates and EGFR21 wild-type homozygous body masterplate, its control systems all can go out goal gene fragment by Successful amplification; And for the object band that the 5th pipe appearance that normal system only has the L747_S752del of EGFR19 to suddenly change the 2nd pipe of heterozygote sample and T790M (6240) the sudden change heterozygote sample of EGFR20 becomes clear, all the other each pipe object bands are all very faint; And for the normal system of EGFR21 homozygote sample, the amplified band of 5 pipes is all very faint; Therefore directly can judge that EGFR21 homozygote sample is as EGFR gene wild-type, and the G719A extracting EGFR18 respectively suddenlys change, the 5th pipe amplified production band of the 1st pipe of heterozygote sample, the 2nd pipe of the L747_S752del sudden change heterozygote sample of EGFR19 and T790M (6240) the sudden change heterozygote sample of EGFR20, reclaims with the agarose gel purification test kit of Sangon Biotech (Shanghai) Co., Ltd..

(5) goal gene fragment sequence measures

Reclaim product for 3 rubber tapping in (4), send Sangon Biotech (Shanghai) Co., Ltd. to check order, peak figure is as shown in Fig. 3 (only representing unidirectional Sequencing chromatogram) in order-checking.As shown in Figure 3, in each heterozygosis system all there is obvious sudden change peak in mutational site place, and sudden change peak is all not less than normal peak; Further analysis finds that GCC in EGFR18 peak figure sports the CGG that GGC(is equivalent to complementary strand and sports CCG), occurred deletion mutantion after GGAA in EGFR19 peak figure, in EGFR20 peak figure, GGT is mutated into the CCA that GAT(is equivalent to complementary strand and sports CTA).

Obviously, above-described embodiment is only for the example done clearly is described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And therefore amplified apparent change or variation are still within the protection domain of the invention.

SEQUENCELISTING

<110> Medical University Of Fujian

<120> to check order the method for Sensitive Detection human EGFR gene mutations and test kit thereof based on Sanger

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gctgggtgcggaagaga17

Claims

1. check order based on Sanger the primer of Sensitive Detection human EGFR gene mutations, it is characterized in that: described primer by amplimer group be connected primer sets and form, the nucleotide sequence of described connection primer sets is as shown in SEQ.IDNO.9-SEQ.IDNO.26.

2., based on a test kit for Sanger order-checking Sensitive Detection human EGFR gene mutations, it is characterized in that: described test kit adds to connect primer sets, heat-resistant dna ligase enzyme and damping fluid thereof as claimed in claim 1 in EGFR gene fragment amplification system.

3. the test kit of the Sensitive Detection human EGFR gene mutations that checks order based on Sanger according to claim 2, is characterized in that: described test kit comprises archaeal dna polymerase, PCR damping fluid, dNTPs, 8 amplimers, 18 connections primer, heat-resistant dna ligase enzyme and heat-resistant dna ligase enzyme damping fluids; Described 8 its sequences of amplimer are as shown in SEQ.IDNO.1-SEQ.IDNO.8, and wherein SEQ.IDNO.1 and SEQ.IDNO.2, SEQ.IDNO.3 and SEQ.IDNO.4, SEQ.IDNO.5 and SEQ.IDNO.6, SEQ.IDNO.7 and SEQ.IDNO.8 are respectively used to the amplification of target the 18th, 19,20,21 exon fragment; Described 18 connect its sequence of primer as shown in SEQ.IDNO.9-SEQ.IDNO.26, wherein 3 kinds of mutational sites of SEQ.IDNO.9 and SEQ.IDNO.10 target the 18th exon, 19 kinds of mutational sites of SEQ.IDNO.11 and SEQ.IDNO.12 target the 19th exon, 5 kinds of mutational sites of SEQ.IDNO.13-SEQ.IDNO.22 target the 20th exon, 2 kinds of mutational sites of SEQ.IDNO.23-SEQ.IDNO.26 target the 21st exon.

4. the test kit of the Sensitive Detection human EGFR gene mutations that checks order based on Sanger according to claim 3, is characterized in that: the enzyme activity unit concentration of described archaeal dna polymerase is 5U/uL, has resistance toheat; Described PCR damping fluid is by 100mmol/LTris-HCl, 500mmol/LKCl and 15mmol/LMgCl of pH=8.3 ₂composition; Described dNTPs is mixed by each 2.5mmol/L of dATP, dTTP, dCTP and dGTP; The concentration of described amplimer is 10umol/L; The concentration of described connection primer is 10umol/L; The enzyme activity unit concentration of described heat-resistant dna ligase enzyme is not less than 4000U/uL, and under normal system, 95 degree of insulation 1h enzyme activities are not less than 60% of protoenzyme work; Described heat-resistant dna ligase enzyme damping fluid be formulated as 20mMTris-HCl, 25mM potassium acetate, 10mM magnesium acetate, 1mMNAD, 10mMDTT, 0.1%TritonX-100, pH=7.6.

5. utilize a method for the Sensitive Detection human EGFR gene mutations that checks order based on Sanger of test kit according to any one of claim 2-4, it is characterized in that the step of described method is as follows:

(1) sample collection and extracting genome DNA: gather or take out biological specimen to be measured, utilizes commercial test kit to extract its genomic dna;

(2) the specific fragment amplification of EGFR gene: the amplification system 5 adopting described test kit to prepare the combination of EGFR gene exon is respectively managed; Often all the other are all identical except being connected primer difference except amplimer for pipe amplification system, and primer comprises following combination: the 1st body system comprises these two pairs of amplimers of SEQ.IDNO.1-SEQ.IDNO.2 with SEQ.IDNO.5-SEQ.IDNO.6 and this two couple of SEQ.IDNO.9-SEQ.IDNO.10 with SEQ.IDNO.13-SEQ.IDNO.14 is connected primer; 2nd body system comprises these two pairs of amplimers of SEQ.IDNO.3-SEQ.IDNO.4 with SEQ.IDNO.5-SEQ.IDNO.6 and this two couple of SEQ.IDNO.11-SEQ.IDNO.12 with SEQ.IDNO.15-SEQ.IDNO.16 is connected primer; 3rd body system comprises SEQ.IDNO.5-SEQ.IDNO.6 this pair amplimer and SEQ.IDNO.17-SEQ.IDNO.18, and this is connected primer a pair; 4th body system comprises these two pairs of amplimers of SEQ.IDNO.7-SEQ.IDNO.8 with SEQ.IDNO.5-SEQ.IDNO.6 and this two couple of SEQ.IDNO.19-SEQ.IDNO.20 with SEQ.IDNO.23-SEQ.IDNO.24 is connected primer; 5th body system comprises these two pairs of amplimers of SEQ.IDNO.7-SEQ.IDNO.8 with SEQ.IDNO.5-SEQ.IDNO.6 and this two couple of SEQ.IDNO.21-SEQ.IDNO.22 with SEQ.IDNO.25-SEQ.IDNO.26 is connected primer; 25uL system is specially to 10 × PCR damping fluid of 2.5uL, the dNTPs of 2-3uL, every bar amplimer 0.5-1uL, every bar connects primer 0.25-0.5uL, 10 × heat-resistant dna ligase enzyme damping fluid of 1-2.5uL, 0.1-0.3uL heat-resistant dna ligase enzyme, 0.1-0.2uLDNA polysaccharase, 20-100ng genomic dna, the final sterilizing deionized water that adds complements to 25uL; Corresponding not the managing containing the contrast amplification system 5 connecting primer, heat-resistant dna ligase enzyme damping fluid and these three kinds of components of heat-resistant dna ligase enzyme of preparation simultaneously, scarce component addition is substituted by sterilizing deionized water, and all the other components are with corresponding normal system; Totally 5 pipes manage containing the normal system and 5 of ligase enzyme system the amplification that the control systems not containing ligase enzyme carries out EGFR gene fragment on gene-amplificative instrament, and amplification condition is: 95 DEG C of denaturation 5-10min; 94 DEG C of sex change 15-30s, 50-53 DEG C of annealing 45-90s, 72 DEG C extend 1-2min, circulation 30-36 time; 72 DEG C of ends extend 10-12min;

(3) rubber tapping of goal gene fragment is reclaimed: all PCR primer carry out agarose gel electrophoresis, and dye 10-20min be placed on clear water decolouring and gel imaging system take pictures and analyzes in 0.5-1ug/mlEB solution; When control systems Successful amplification goes out goal gene fragment, with DNAMarker band for standard, select the band that in normal system, each exon amplification production concentration is not less than 10ng/uL to tap rubber, and reclaim with commercial gel purification kit;

(4) goal gene fragment sequence measures

Product is reclaimed for rubber tapping, carries out two-way order-checking and analysis, according to the sequence of all order-checking peak figure final analysis goal gene fragments according to the standard operation of DNA fragmentation order-checking;

(5) EGFR sudden change result judges and application

When control systems normally increases, if each exon amplification band in normal system is all without the object band meeting rubber tapping recovery, then reclaiming and subsequent operations without the need to carrying out rubber tapping, directly judging that the EGFR gene detected result of sample is as feminine gender and wild-type; If there is the object band meeting rubber tapping and reclaim, and two sequencing result is shown as the corresponding known mutations connecting primer target, then judge that the EGFR gene detected result of sample is as the positive and saltant type, mutation type is as the criterion with the actual known mutations measured; If two-way sequencing result is same unknown difference site, then need to judge that the EGFR gene detected result of sample is as new mutant/polymorphism further, sudden change/polymorphism type with actual measure be as the criterion; If two-way sequencing result is wild-type, then judge that the EGFR gene detected result of sample is as judging, needs to redeterminate; If two-way sequencing result is one to for wild-type one is to being saltant type, then judge that the EGFR gene detected result of sample is as judging, needs to redeterminate.

6. the method for the Sensitive Detection human EGFR gene mutations that checks order based on Sanger according to claim 5, it is characterized in that described connection primer SEQ.IDNO.9, SEQ.IDNO.11, SEQ.IDNO.13, SEQ.IDNO.15, SEQ.IDNO.17, SEQ.IDNO.19, SEQ.IDNO.21, SEQ.IDNO.23, 5 ' the end of SEQ.IDNO.25 and 3 ' first Nucleotide held all carry out phosphorylation modification, SEQ.IDNO.10, SEQ.IDNO.12, SEQ.IDNO.14, SEQ.IDNO.16, SEQ.IDNO.18, SEQ.IDNO.20, SEQ.IDNO.22, SEQ.IDNO.24, first Nucleotide that 3 ' of SEQ.IDNO.26 holds all carries out phosphorylation modification.