CN102533958A - Method and kit for detecting human PIK3CA gene mutation - Google Patents
Method and kit for detecting human PIK3CA gene mutation Download PDFInfo
- Publication number
- CN102533958A CN102533958A CN2010106085005A CN201010608500A CN102533958A CN 102533958 A CN102533958 A CN 102533958A CN 2010106085005 A CN2010106085005 A CN 2010106085005A CN 201010608500 A CN201010608500 A CN 201010608500A CN 102533958 A CN102533958 A CN 102533958A
- Authority
- CN
- China
- Prior art keywords
- pik3ca
- exon
- pcr
- seconds
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a method and kit for detecting gene mutation, and specifically relates to a method and kit for detecting PIK3CA gene mutation. The invention is characterized in that the kit comprises two specific probes for performing genotyping on a 9<th> exon and a 20<th> exon of the PIK3CA gene, wherein the specific probe for the 9<th> exon contains a nucleotide sequence of E545K and E542K codons, and the specific probe for the 20<th> exon contains a nucleotide sequence of an H1047R codon. By using the conventional PCR (polymerase chain reaction) amplification in combination with the Cold-PCR amplification product enrichment technology and the high-resolution melting curve analysis technology, the kit provided by the invention can complete the judgment on the genotyping of a sample.
Description
Technical field
The present invention relates to biotechnology and medical field, be specifically related to a kind of method and test kit of the PIK3CA of detection transgenation.
Background technology
The PIK3CA gene is that retrovirus v-p3k oncogene is at intracellular homologue; I class phosphatidyl 3 kinases (the phosphatidylinositol 3-kinases of coding; PI3Ks) by a p110 catalytic subunit and the different first dimer that the p85 regulator subunit is formed; By growth factor receptor tyrosine kinase such as EGF-R ELISA (epidermal growth factor receptor, EGFR), activation such as insulin receptor.Catalysis PI 4 phosphoric acid (PI-4-P) and PI 4 after the PI3Ks activation, the 5-di-phosphate (PI-4,5-P2) phosphoric acid change into PI 3,4 di-phosphate (PI-3,4-P2) with PI 3,4,5 triphosphoric acids (PI-3,4,5-P3, Ptd InsP3).Thereby a series of albumen of activation; Comprise and regulate cell proliferation, survival and cell cycle regulating etc., relate to numerous target molecules such as mTOR (target of repamycin), BAD, Caspace 9, Tuberin, GSK3 β and FH (Fork head) transcription factor family subgroup.That wherein important is Soviet Union-serine kinase AKT.AKT be positioned to plasma membrane after Ptd Ins P3 combines, then by phosphoinositide deopendent protein kinase 1 and 2 activation of phosphoinositide deopendent protein kinase, thereby suppress apoptosis, promote cell survival.When the PIK3CA gene activated with forms such as gene amplification or point mutation, it was transcribed with translation skill and raises, and through the PI3K-AKT approach, makes the AKT excessive activation, unconfined promotion cell growth and breeding, and suppress apoptosis, finally cause tumour to take place.
Kinase domain exons coding district to whole 16 members of PI3K family carries out the sequential analysis demonstration, and PIK3CA is that unique coming to light causes carcinogenic gene by somatic mutation.The sudden change 80%-90% that a plurality of researchs all propose the PIK3CA gene accumulates in the 9th exon and these two hot spot regions of the 20th exon of this gene, respectively to helical region and kinases district that should enzyme.With the cell in vitro cultured method to these two focus saltation zones discover apoptosis that its sudden change not only can reduce cell can also promote tumour infiltration, improve the activity of its downstream kinases P13Ks.Discover that about these two hot zones of PIK3CA sudden change the sudden change of kinases district and helical region possibly cause the functional change of enzyme through different mechanism.The sudden change of different zones, the activation that causes P13Ks through regulator subunit p85 and the interactional different mechanisms of RAS-GTP respectively with P13Ks.Helical region sudden change does not rely on when inducing acquired changing function and the combining of p85, but need with the interaction of RAS-GTP; Opposite, the sudden change in kinases district is effectively when lacking RAS-GTP, but highly relies on p85.It is thus clear that the sudden change of kinases district and helical region is that the sudden change of these two kinds of mechanism is collaborative each other in same molecule through two different and independently machine-processed playing a role.
Research shows that PIK3CA amplification occurs or crosses and express in kinds of tumors.The research report is arranged, and the high frequency somatic mutation takes place in the PIK3CA gene in kinds of tumors such as colorectal carcinoma, ovarian cancer and mammary cancer, cancer of the stomach and liver cancer.There is research to confirm that the PIK3CA transgenation is one of modal transgenation in the mammary cancer.The frequency of PIK3CA transgenation is at 8%-40% in the breast cancer tissue.Research recently confirms the activation of PI3K path, no matter is through the activation of inactivation or the oncogene PIK3CA of cancer suppressor gene PTEN, all can cause the resistance of patient with breast cancer to targeted drug Herceptin.
In the 35th European Internal Medicine-Oncology association (ESMO) conference in 2010 10, announced prognosis and the outcome prediction Study on Value result of a relevant PIK3CA sudden change to EGF-R ELISA-tyrosine kinase inhibitor (EGFR-TKI) treatment nonsmall-cell lung cancer (NSCLC).PIK3CA, EGFR, the KRAS mutation rate of going into to organize the patient are respectively 4.8%, 25.3%, 6.8%.PIK3CA sudden change with alleviate uncorrelated (P=0.96), but with short to the PD time (TTP, P=0.04) and the OS phase (P=0.005) relevant.EGFR suddenly change well relevant with curative effect (P＜0.0001).The KRAS sudden change is relevant with short TTP (P=0.003), but irrelevant with OS.Multivariate analysis shows, proofread and correct histological type and physical state (PS) after, the PIK3CA sudden change is still the remarkable predictive factors (P=0.004) of OS, KRAS, PIK3CA sudden change still indicate TTP weak point (P=0.009, P=0.003).The investigator thinks and should KRAS, PIK3CA sudden change be included in EGFR-TKI treatment resistance and the existence prediction indication thing.
Kato etc. (Kato S, Iida S, Higuchi T, et al.2007) carry out PIK3CA sudden change and KRAS mutation analysis to 158 routine CRC tissue samples, and the result shows that the PIK3CA mutation rate is 11.4%, and the KRAS mutation rate is that 31.6%, two kind of sudden change does not have dependency.The PIK3CA sudden change is considered to the independent prognostic factor that II/III phase patient does not have recurrence existence.Recent research has shown the predicting function of this pathway activity to the Cetuximab therapeutic response; (Frattini M, Saletti P, Romagnani E such as Frattini; Et al.2007) finds; The mCRC patient of 27 routine EGFR high expression levels is after the Cetuximab treatment, and PTEN normal expression person part remission rate is 62.5%, and PTEN disappearance person is all invalid.(Sartore-Bianchi A such as nearest Sartore-Bianchi; Martini M; Molinari F, et al.2009) report in the 109 example colorectal cancer patients in late period, KRAS, BRAF wild-type 77 examples (71%), wherein 20 examples are effective to the treatment of associating Cetuximab or handkerchief Buddhist nun monoclonal antibody.Have 59 examples and successfully accepted the detection of PIK3CA and PTEN gene, in 27 examples of PIK3CA transgenation or PTEN expression deletion, 1 example is only arranged effectively, and other 32 examples do not have sudden change or (with) among the patient of expression deletion, 17 examples are arranged effectively.Multivariate analysis shows, the PIK3CA transgenation and lifetime significant correlation.PIK3CA transgenation colorectal cancer patients is accepted anti-EGFR monoclonal antibody unsatisfactory curative effect, and KRAS and PIK3CA gene joint-detection be predict anti EGFR monoclonal antibody curative effect better.More than find prompting,, before treatment, carry out the detection that KRAS/BRAF, PI3KCA sudden change and PTEN express situation, possibly select the EGFR targeted therapy positive meaning to be arranged individuation to avoid unnecessary toxicity and economical load for tumour patient.
Current, traditional direct sequence measurement is " gold standard " that carries out the PIK3CA detection in Gene Mutation both at home and abroad always.Direct sequencing has directly and advantage intuitively; The site and the type of understanding PIK3CA transgenation that can be definite; But the sensitivity of this method is low, just can detect when requiring mutation rate to reach 20%-30%, therefore needs micro-dissections or selectivity to organize extracting to come the enrichment mutant cell.And expense is high, flux is low, sense cycle is long, and the clinical application limitation is big.Pvuii restriction fragment is analyzed (RFLP) though have higher sensitivity, and the mutational site of being detected must have the restriction enzyme site of restriction enzyme, actual design and complicated operation.
The sensitivity of efficient sex change liquid phase (dHPLC) but can only be distinguished the heterozygous sample between 5%-10%, when detecting homozygous sample, need mix the wild-type sample mixing and become heterozygous to detect, and brings extra testing cost, has prolonged sense cycle.
The technology of state-of-the-art in the world detection PIK3CA transgenation is to adopt site-specific quantitative fluorescent PCR; The advantage of this method has been to improve the ageing of detection sensitivity and detection, but the real-time fluorescence quantitative PCR detection technique itself exists the problem of false positive rate height that is poor specificity.
Several kinds of top detection methods are owing to the defective of self, and limitation is bigger aspect clinical application, so far still just as a kind of detection method, but not the finished product test kit.
It is an emerging technology that high resolving power fusion (High-resolution melting is called for short HRM) is analyzed.It only through the melting curve analysis after the PCR, just can detect the segmental small sequence difference of PCR, thereby is applied in many aspects such as sudden change scanning, sequence pairing and gene type.HRM both can carry out examination, scanning to unknown mutation, also can analyze known mutations.For traditional mutation analysis, operation steps is simplified greatly, and time and cost have also reduced much.And sample directly carries out HRM and analyzes behind pcr amplification, need not to shift, and really realized the stopped pipe operation, reduced the risk of polluting.
In the prior art, adopt the report of high resolving power liquation PIK3CA transgenation to have:
1, human such as Panagiotis A.Vorkas is through to known 129 paraffin-embedded breast tissue samples; Wherein 10 of 99 of tumor specimens, 20 of non-cancer samples, fibroma sample detect the sudden change situation of PIK3CA the 9th and No. 20 exons with the PCR-HRM method and detected result and dna sequencing result are compared; The result shows that No. 9 exons of 12/99 (12.1%) tumor specimen PIK3CA gene undergo mutation, and the mutation rate of No. 20 exons is 20.2% (20/99).In non-cancer sample, do not find the situation of PIK3CA transgenation, the PIK3CA transgenation of 1 part of fibroma sample.The detected result of PCR-HRM method is consistent with dna sequencing, good reproducibility, and sensitivity can reach 1%.They have detected 75 with the PCR-HRM method and have accepted the patient with breast cancer PIK3CA gene the 9th of trastuzumab treatment and the sudden change situation of 20 exons then, and the mutation rate of PIK3CA reaches 40% (30/75).The result proves with the PCR-HRM method and detects the highly sensitive of PIK3CA transgenation, good reproducibility, and it is low, simple to operate to detect cost, can be implemented in the high throughput testing under the large sample situation, and clinical value is preferably arranged.(reference: Panagiotis A.Vorkas.Nikoleta Poumpouridou, Journal of MolecularDiagnostics 2010, Vol.12, No.5)
2, Suzhou is that a kind of PIK3CA transgenation test kit is sold by true biological medicine ltd; Adopt the analysis of high resolving power melting point curve; Can go up and analyze being furnished with the quantitative fluorescent PCR instrument of HRM module (like LightCylcer480 etc.); Be used to detect two focus saltation zones of PIK3CA gene: one is E542K and the E545K sudden change that is positioned at No. 9 exons, and another is the H1047R sudden change that is positioned at No. 20 exons.Test kit can be used for fresh or alcohol fixation, paraffin-embedded specimens from pri, also can be used for the detection of the non-specimens from pri such as puncture or biopsy specimen, blood preparation, stool sample of trace.
Though existing test kit has adopted the method for HRM, in reality, often need subsequent P CR enrichment and dna sequencing to come gene type is carried out in the sudden change that detects, this is because the gene mutation site of PIK3CA is more, and gene type is carried out in sudden change.There is research to show that No. 9 exons mutations of PIK3CA gene do not influence the anti-curative effect in western appropriate Xidan; No. 20 exons mutation then indicates prognosis mala; Therefore can help clinical analysis and scientific research with effectively making a distinction to the accurate identification in mutational site with the prognosis of tumour and the closely-related sudden change of targeted therapy curative effect and other sudden changes.
Summary of the invention
The object of the invention provides a kind of test kit that contains the detection people PIK3CA transgenation of specific probe; Remedy HRM and detect the shortcoming that to carry out Mutation Screening; Realize gene type; Through optimization to detection reagent and testing conditions, make this test kit simple to operate fast, detected result is more stable, the detection cost is also lower.
For achieving the above object; The technological method that the present invention adopts is: the test kit of a kind of people of detection PIK3CA transgenation; It is characterized in that said test kit comprises two specific probes that are used for No. 9 exon of PIK3CA gene and No. 20 exon are carried out gene type; The specific probe of said No. 9 exon comprises the nucleotide sequence of E545K and 542K codon; The specific probe of No. 20 exon comprises the nucleotide sequence of H1047R codon, and No. 9 exon of said PIK3CA has the continuous nucleotide sequence of SEQ IDNo:1; No. 20 exon of said PIK3CA has the continuous nucleotide sequence of SEQ ID No:2.
The specific probe of described No. 9 exon is SEQ ID No.3, and the specific probe of No. 20 exon is SEQ ID No.4.
Described specific probe 3 ' is held through sealing treatment, can't be extended.
Described test kit also comprises PCR damping fluid, dNTP, Taq archaeal dna polymerase, Auele Specific Primer to, SYTO 9 optical dyes, water and wild-type contrast, and wherein Auele Specific Primer is selected from:
Said reaction system; Per 20 μ l PCR reaction system final concentrations consist of: 2 μ l PCR damping fluids (10 *), 0.6-1.0 μ l magnesium chloride solution, 1.6 μ l dNTP, 0.1-0.3 μ l Taq archaeal dna polymerase, 100-200nM forward primer and 20-40nM reverse primer, 100-200nM specific probe; 2-5 μ l SYTO 9 optical dyes, all the other are water.
In the technique scheme, said TaqDNA polysaccharase has the warm start characteristic, and vigor is 5 every microlitres of unit; Said DNA optical dye is saturated fluorescence dyestuff SYTO 9, and concentration is 50 μ M.
In the technique scheme, 10 * PCR damping fluid (multiple of dilution when numeral 10 expressions are used), by TrisCl, KCl, (NH
4)
2SO
4, 15mM MgCl
2Form, pH 8.7.
In the technique scheme, described mM and μ M are the units of volumetric molar concentration, refer to the mole number of contained solute in every liter of solution.
In the technique scheme, described SYTO 9 is highly sensitive DNA optical dyes, can combine with double-stranded DNA, and reaction does not still have restraining effect to PCR under the situation of high density.
The mentioned reagent box can be measured the genomic dna in people source, sample not restriction as, body fluid (comprising blood, ascites), histocyte (tumor tissues) etc. are through extracting and these samples of purifying can prepare genomic dna.
In the technique scheme, from genomic dna, amplification contains the dna fragmentation of PIK3CA E542K and E545K codon gene, and to obtain being used to measure melting curve, designed primer is arranged in people PIK3CA gene extron 9, and concrete sequence is SEQ ID No:1.
In the technique scheme, from genomic dna, can increase contains the dna fragmentation of PIK3CA H1047R codon gene, and to obtain being used to measure melting curve, designed primer is arranged in people PIK3CA gene extron 20, and concrete sequence is SEQ ID No:2.
In the technique scheme, contain the sample that the dna fragmentation of PIK3CA gene E542K, E545K and H1047R codon obtains, be particularly suitable for as measuring material through amplification.For example, can use primer to increase by PCR method, this primer contains the part of PIK3CA gene E542K, E545K and H1047R codon through design rationally so that only increase.Auele Specific Primer of the present invention is one of inventive point of the present invention, and this primer those skilled in the art often regulation are equipped with the method preparation of primer.The base length in district to be amplified is unrestricted, but recommends the fragment less than 150 bases.When by preparation primer according to the invention, the working sample that can obtain to suit, it is as the dna fragmentation of amplification, and has specificity length.
Another object of the present invention is the method for No. 9 exon of a kind of PIK3CA of detection gene and No. 20 exons mutation and gene type, its characteristic with said method comprising the steps of:
1) gathers genomic dna to be analyzed according to ordinary method;
2) said gene group DNA is carried out the pcr amplification of PIK3CA gene 9 and No. 20 exons respectively, again the pcr amplification product that obtains is carried out enrichment with Cold-PCR; Reaction becomes renaturation with amplified production after accomplishing again;
3) above-mentioned amplified production is carried out the melting curve analysis, the operation melting curve writes down out peak temperature, and operation Genescan auto-analyzer procedure is through comparing software automatic distinguishing wild-type and mutant with the wild-type contrast.After confirming that through Genescan software the sample gene is undergone mutation; Little probe binding sequence in the melting curve of comparative sample and positive control go out peak temperature; If both go out the peak temperature unanimity; Then show sample at No. 9 exon 542,545 codons of PIK3CA, 1047 codon places of No. 20 exon go out to undergo mutation; If both to go out peak temperature inconsistent, then show sample at No. 9 exon 542,545 codons of PIK3CA, the site producer sudden change beyond No. 20 exon 1047 codons.
In the test kit of detection PIK3CA of the present invention transgenation, sample to be tested is diluted to same concentration, do the wild-type contrast simultaneously; Add in the test kit; Carry out a PCR reaction, the operation melting curve carries out data analysis with Genescan software; Can distinguish wild-type and mutant, and gene type is carried out in the sudden change that detects.The present invention is applicable to the sudden change detection and the gene type of No. 1047 codon of the 542nd codon and 545 codons and No. 20 exon of No. 9 exon of PIK3CA gene.
Wherein, step 2) in, described primer is to being the following table sequence:
Wherein, step 2) in, described characteristic probe sequence is:
| No. 9 exons | Specific probe | SEQ?ID?No：3 |
| No. 20 exons | Specific probe | SEQ?ID?No：4 |
The pcr amplification system of per 20 μ l comprises: genomic dna solution, 2 μ l PCR damping fluids (10 *), 0.6-1.0 μ l magnesium chloride solution, 1.6 μ l dNTP, 0.1-0.3 μ l Taq archaeal dna polymerase, 100-200nM forward primer and 20-40nM reverse primer, 100-200nM specific probe; 2-5 μ lSYTO 9 optical dyes, all the other are water.
The process of pcr amplification is: 95 ℃ 15 minutes, 95 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 20 circulations.
The process of Cold-PCR enrichment is: 82 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, 15 circulations, 72 ℃ 2 minutes.
After reaction is accomplished, the PCR product is become renaturation again, 95 ℃ 30 seconds, 50 ℃ 1 minute.
The volume of the genomic dna solution that adds in the technique scheme, step 2) is adjusted according to DNA concentration in the extracting solution, makes the DNA amount of adding reach 10ng.
In the technique scheme, in the step 3), the pcr amplification product sample carries out the melting curve analysis in LightCycler 480 appearance.Instrument is warmed up to 95 ℃, the melting curve of acquisition sample with the speed of 0.2 ℃/s from 72 ℃.
Cardinal principle of the present invention is:
1) adopt the HRM technology before the PCR reaction, to add the saturated fluorescence dyestuff, in certain temperature range, pcr amplification product is carried out sex change, the dna double chain is unwind gradually, this moment, luminescent dye molecule came off from the dna double chain gradually, and fluorescent signal descends.After PIK3CA codon E542K, E545K and H1047R undergo mutation; The melting temperature (Tm) (Tm value) of the PCR product of process primer amplified can change; LightCycler 480 is through the optical detection fluorescent signal and draw the temperature melting curve, accurately distinguishes wild-type and mutant according to curve.
2) COLD-PCR is a new low abundance sudden change beneficiation technologies; The maximum difference of it and conventional PCR is in the PCR process, to have adopted crucial denaturation temperature (Tc); This Tc is lower than the denaturation temperature of standard, therefore under the situation of Tc temperature, has only heteroduplex sex change to unwind and to increase with the primer pairing subsequently; The higher homoduplex of denaturation temperature then can not unwind, thereby can not increase.After several circulations, the mutated genes group has obtained advantage pcr, and proportion increases in total genome, has obtained the purpose of sudden change enrichment.Because initial template amount is less, we select to have adopted conventional PCR and COLD-PCR bonded technological line, and the standard denaturation temperature of using is earlier carried out the conventional PCR of 20 round-robin; And then, the crucial denaturation temperature of employing is carried out 15 round-robin COLD-PCR.
3) specific probe combines the HRM technology, on the Mutation Screening basis, can carry out gene type.Specific probe has comprised PIK3CA codon E542K, E545K and H1047R, and complementary with amplified production.In the PCR system, we have adjusted the concentration of reverse primer, and its concentration is 1/5th of forward primer, and such asymmetric PCR can amplify the PCR product of forward primer in a large number, and a small amount of reverse primer product that only increases.At the fusion initial stage; Specific probe and a large amount of strand forward primer product complementary pairings are thus in fusion processes, along with two sections glimmering melting curves can successively appear in the rising of temperature; One section is caused by specific probe, and another section then is that conventional double-stranded PCR product causes.When the melting curve of the double-stranded PCR product of routine was shown as mutant, we can confirm whether sudden change occurs on codon E542K, E545K and the H1047R through the melting curve of specific probe, or occur in the new mutant of other positions.
Therefore, the present invention is through the primer of above-mentioned optimization, according to the PCR method required dna fragmentation that increases.The optical detection fluorescent signal changes generation temperature melting curve and confirms that they show different melting curves.According to the melting curve that in aforesaid method, obtains, can measure the sudden change situation of PIK3CA the 9th and No. 20 exons.
Because the technique scheme utilization, the present invention compared with prior art has advantage:
1, codon E542K provided by the invention, E545K and H1047R sudden change detection kit; What adopt is a kind of biological detection method; Compare with existing detection kit, have obvious improvement, do not have special limitation for sample like test kit of the present invention; No matter be that body fluid or cell all can be used as detection sample of the present invention, make easy to detect quick.
2, the Auele Specific Primer of the present invention design base length of treating amplification region does not have strict demand, can obtain PCR product of the present invention and carry out the analysis of high resolving power melting curve through Auele Specific Primer of the present invention.
3, the present invention has added specific probe in the PCR reaction system, and through the asymmetric PCR reaction, codon mutation and other mutational sites in the amplified fragments that can known mutation frequency is the highest be effectively distinguished, and gene type is more accurate.
4, the present invention has compared with prior art introduced the COLD-PCR beneficiation technologies, and is easy and simple to handle, detects with low costly, and detected result is accurate, and sensitivity has well using value and marketable value when participating in the cintest up to 0.1%-0.05%.
Accompanying drawing
9 extras show sub-wild-type, homozygous mutation and three kinds of genotypic melting curves of heterozygous mutant among Fig. 1 embodiment two;
20 extras show sub-wild-type, homozygous mutation and three kinds of genotypic melting curves of heterozygous mutant among Fig. 2 embodiment two;
Detect the sensitivity test of No. 9 exons among Fig. 3 embodiment three
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one: the extraction of choice of sample and genomic dna
The genomic dna of collecting derives from whole blood, cell, flesh tissue and paraffin fixing organization respectively, and the operational manual that process for extracting reference reagent box provides has been done simultaneously partly and optimized.
The healthy subjects Whole Blood Genomic DNA is used the BloodGen Mini Kit extraction of health as century as the wild-type contrast of experiment;
The genomic dna that derives from the OMC-1 cell strain as No. 9 exon homozygous mutation type of PIK3CA gene of experiment (1633G＞A) contrast,
(1633G＞A) contrast uses the DNA FlexGen DNAKit extraction of health as century to the genomic dna that derives from the MCF-7 cell strain as No. 9 codon heterozygous mutant type of PIK3CA gene of experiment;
The genomic dna that derives from the SK-OV-3 cell strain as No. 20 exon homozygous mutation type of PIK3CA gene of experiment (3140A＞G) contrast,
(3140A＞G) contrast uses the DNA FlexGen DNAKit extraction of health as century to the genomic dna that derives from the HCT-116 cell strain as No. 20 codon heterozygous mutant type of PIK3CA gene of experiment;
And flesh tissue and the paraffin organization mouse of all originating is used to assess the flexibility of HRM method to fixing organization, uses health to extract as the DNA TissueGen DNA Kit in century.
The tissue-derived sudden change situation of cell strain genotype amino acid
OMC-1 cervical cancer 1633G＞A homozygote E545K
MCF-7 mammary cancer 1 633G＞A heterozygote E545K
SK-OV-3 ovarian cancer 3140A＞G homozygote H1047R
HCT-116 colorectal cancer 3140A＞G heterozygote H1047R
Genomic dna all meets laboratory Quality Control requirement: OD260/280 is between 1.8-2.0; Electrophoresis result shows that except that the paraffin fixing organization, all the other genomic DNA fragments all are the above big fragments of 10kb, and there is the part degraded in the genomic dna of paraffin fixing organization, and fragment size distribution is between 1000-23kb.
Dna solution concentration is adjusted to the every microlitre of 10ng, and-20 ℃ of preservations are subsequent use.
Embodiment two: the identification in mutational site
Adopt the HRM technology that No. 9 exon E542K of known PIK3CA gene, E545K codon and No. 20 genotypic sample of exon H1047R codon are detected.
1. the Auele Specific Primer and the probe of No. 9 exon are following:
SEQ?ID?No：5，Forward?primer：
5’-CAGCTCAAAGCAATTTCTACACGAGAT-3’
SEQ?ID?No：6，Reverse?primer：5’-TTAGCACTTACCTGTGACTCCATAGA-3’
SEQ ID No:3, specific probe: 5 '-TCTTTCTCCTGCTCAGTGAT-3 '
The Auele Specific Primer and the probe of No. 20 exon are following:
SEQ?ID?No：11，Forward?primer：5’-GCAAGAGGCTTTGGAGTATTTCAT-3’
SEQ?ID?No：12，Reverse?primer：5’-TGCTGTTTAATTGTGTGGAAGATCC-3’
SEQ ID No:4, specific probe: 5 '-CCACCATGATGTGCATCATTC-3 '
2. through near the PCR part fragment No. 9 exon E542K, E545K codon and No. 20 exon H1047R codons that increases respectively; The preparation mixed solution: each 0.08 μ l of genomic dna solution 1 μ l, 2 μ l PCR damping fluids (10 *), 1.6 μ l dNTP, 0.1 μ l Taq archaeal dna polymerase, forward primer 0.4 μ l and the reverse primer of preparation, SYTO 9 optical dyes 2 μ l before adding, adding other pure water of PCR level, to make reaction volume be 20 μ l.Be reflected at 95 ℃ 15 minutes, 95 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 20 circulations, 82 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 15 circulations, 72 ℃ 2 minutes; After reaction is accomplished, the PCR product is become renaturation again, 95 ℃ 30 seconds, 50 ℃ 1 minute;
3. through sample is carried out the melting curve analysis in LightCycler 480 appearance.Instrument is warmed up to 95 ℃ with the speed of 0.2 ℃/s from 72 ℃, obtains the melting curve of sample, and as shown in Figure 1, Fig. 1 is No. 9 exon somatotype data plots; Melting curve is per sample distinguished genotype; As shown in Figure 2, Fig. 2 is No. 20 exon somatotype data plots; Melting curve is per sample distinguished genotype
The foregoing description result verification:
The homozygous mutation and the heterozygous mutant sample of No. the 9th, PIK3CA gene and No. 20 exons can be effectively discerned in experiment.
Embodiment three: the checking of test kit susceptibility
With No. 9 exons of PIK3CA gene is example, and the wild type gene group of extracting in the OMC-1 genome of homozygous mutation type and the whole blood is mixed by a certain percentage, makes homozygous mutation type genome account for 0.05% of total DNA amount.Above-mentioned mixing genomic dna and wild type gene group DNA concentration are adjusted into the every microlitre of 10ng.
1. the Auele Specific Primer and the probe of No. 9 exon are following:
SEQ?ID?No：5，Forward?primer：
5’-CAGCTCAAAGCAATTTCTACACGAGAT-3’
SEQ?ID?No：6，Reverse?primer：5’-TTAGCACTTACCTGTGACTCCATAGA-3’
SEQ ID No:3, specific probe: 5 '-TCTTTCTCCTGCTCAGTGAT-3 '
2. through near part fragment No. 9 exon E542K of pcr amplification, the E545K codon; The preparation mixed solution: each 0.08 μ l of genomic dna solution 1 μ l, 2 μ l PCR damping fluids (10 *), 1.6 μ l dNTP, 0.1 μ l Taq archaeal dna polymerase, forward primer 0.4 μ l and the reverse primer of preparation, SYTO 9 optical dyes 2 μ l before adding, adding other pure water of PCR level, to make reaction volume be 20 μ l.Be reflected at 95 ℃ 15 minutes, 95 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 20 circulations, 82 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 15 circulations, 72 ℃ 2 minutes; After reaction is accomplished, the PCR product is become renaturation again, 95 ℃ 30 seconds, 50 ℃ 1 minute;
3. through sample is carried out the melting curve analysis in LightCycler 480 appearance.Instrument is warmed up to 95 ℃ with the speed of 0.2 ℃/s from 72 ℃, obtains the melting curve of sample, and as shown in Figure 3, Fig. 3 is No. 9 exon somatotype data plots; Melting curve is per sample distinguished genotype.
The foregoing description interpretation of result:
By the classifying method of mentioning among the embodiment 2, test kit can clearly detect the sample that contains 0.05% homozygous mutation.Concrete genotype is as shown in Figure 3, and present embodiment explanation traditional P CR-HRM technology is after introducing COLD-PCR, and the susceptibility of test kit greatly improves, and reaches as high as 0.05%.
Embodiment four: detection kit
Test kit of the present invention is made up of following reagent, originates as follows, and test kit of the present invention supplies 10 person-portions to detect and uses-20 ℃ of preservations:
| Component | Volume (μ l) | The source |
| PCR damping fluid (10 *) | ?25 | Qiagen |
| DNTP mixed solution (every kind of base 2.5mM) | ?20 | Takara |
| MgCl2(25mM) | ?10 | Qiagen |
| FO primer (10 μ M) | ?6 | Self-control |
| RE primer (2 μ M) | ?6 | Self-control |
| Specific probe (10 μ M) | ?6 | Self-control |
| Taq archaeal dna polymerase (5U/ μ l) | ?1.5 | Qiagen |
| SYTO 9 saturated fluorescence dyestuffs (50 μ M) | ?25 | Invitrogen |
Claims (10)
1. test kit that detects people PIK3CA transgenation; It is characterized in that described test kit comprises two specific probes that are used for No. 9 exon of PIK3CA gene and No. 20 exon are carried out gene type; The specific probe of said No. 9 exon comprises the nucleotide sequence of E545K and 542K codon; The specific probe of No. 20 exon comprises the nucleotide sequence of H1047R codon, and No. 9 exon of said PIK3CA has the continuous nucleotide sequence of SEQ ID No:1; No. 20 exon of said PIK3CA has the continuous nucleotide sequence of SEQ ID No:2.
2. the specific probe of No. 9 exon according to claim 1 is SEQ ID No.3, and the specific probe of No. 20 exon is SEQ ID No.4.
3. specific probe 3 ' according to claim 1 is held through sealing treatment, can't be extended.
4. detection people PIK3CA test kit according to claim 1 also comprises PCR damping fluid, dNTP, Taq archaeal dna polymerase, Auele Specific Primer to, SYTO 9 optical dyes, water and wild-type contrast, and wherein Auele Specific Primer is selected from:
5. the test kit of detection people PIK3CA according to claim 1 transgenation; It is characterized in that per 20 μ l PCR reaction system final concentrations consist of: 2 μ l PCR damping fluids (10 *), 0.6-1.0 μ l magnesium chloride solution, 1.6 μ l dNTP, 0.1-0.3 μ l Taq archaeal dna polymerase, 100-200nM forward primer and 20-40nM reverse primer, 100-200nM specific probe; 2-5 μ l SYTO 9 optical dyes, all the other are water.
6. method that detects people PIK3CA transgenation is characterized in that may further comprise the steps:
1) gathers genomic dna to be analyzed according to ordinary method;
2) said gene group DNA is carried out the pcr amplification of PIK3CA gene 9 and No. 20 exons respectively, again through Cold-PCR enrichment amplified production; Reaction is carried out denaturation melting with amplified production after accomplishing again;
3) according to high resolving power melting curve method the PIK3CA gene 9 after increasing and No. 20 exons are carried out the mutational site detection.
7. method according to claim 6 is characterized in that said step 2) in the process of pcr amplification be: 95 ℃ 15 minutes, 95 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 20 circulations.
8. method according to claim 6 is characterized in that said step 2) in the Cold-PCR enrichment process be: 82 ℃ 15 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, carry out 15 circulations; 72 ℃ 2 minutes; After reaction is accomplished, amplified production is become renaturation again, 90 ℃ 30 seconds, 50 ℃ 1 minute.
9. it is characterized in that step 2 according to claim 6 is said) in the volume of the DNA extraction liquid that adds adjust according to DNA concentration in the extracting solution, make the DNA amount of adding reach 10ng.
10. method according to claim 5 is characterized in that, in the step 3), the pcr amplification product sample carries out the melting curve analysis in LightCycler 480 appearance, and instrument is warmed up to 95 ℃, the melting curve of acquisition sample with the speed of 0.2 ℃/s from 72 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106085005A CN102533958A (en) | 2010-12-28 | 2010-12-28 | Method and kit for detecting human PIK3CA gene mutation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106085005A CN102533958A (en) | 2010-12-28 | 2010-12-28 | Method and kit for detecting human PIK3CA gene mutation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102533958A true CN102533958A (en) | 2012-07-04 |
Family
ID=46341984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010106085005A Pending CN102533958A (en) | 2010-12-28 | 2010-12-28 | Method and kit for detecting human PIK3CA gene mutation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102533958A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816839A (en) * | 2012-07-06 | 2012-12-12 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene |
| CN102851368A (en) * | 2012-10-18 | 2013-01-02 | 广州达健生物科技有限公司 | PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method |
| CN103013993A (en) * | 2012-12-26 | 2013-04-03 | 武汉康圣达医学检验所有限公司 | Primer and method for mass spectrometric detection of hotspot mutation of PIK3CA genes by using primer |
| CN104593515A (en) * | 2015-02-06 | 2015-05-06 | 中国医科大学 | PCR-RFLP method for detecting H-site mutation of PIK3CA gene |
| CN105969875A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination |
| JP2017523810A (en) * | 2014-08-07 | 2017-08-24 | ファーマシスト リミテッドＰｈａｒｍａｓｓｉｓｔ Ｌｔｄ | Method for determining PIK3CA mutation status in a sample |
| CN107254529A (en) * | 2017-06-27 | 2017-10-17 | 郴州市第人民医院 | The kit and method of PIK3CA gene mutations in a kind of detection plasma DNA |
| CN107574244A (en) * | 2016-07-05 | 2018-01-12 | 汪建平 | Detect primer pair, kit and the method for No. 1047 codon mutations of exon of PIK3CA genes 20 |
| CN107893109A (en) * | 2017-11-08 | 2018-04-10 | 重庆邮电大学 | A kind of low abundance gene mutation enrichment method based on removal wild-type sequence |
| CN108004317A (en) * | 2017-11-09 | 2018-05-08 | 上海赛安生物医药科技股份有限公司 | PIK3CA detection in gene mutation system and its kit |
| CN111363827A (en) * | 2020-04-30 | 2020-07-03 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting PIK3CA gene mutation and application method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101445832A (en) * | 2008-12-23 | 2009-06-03 | 广州益善生物技术有限公司 | PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof |
-
2010
- 2010-12-28 CN CN2010106085005A patent/CN102533958A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101445832A (en) * | 2008-12-23 | 2009-06-03 | 广州益善生物技术有限公司 | PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| IRENE ET AL: "The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer", 《JOURNAL OF MOLECULAR DIAGNOSTICS》, vol. 12, no. 5, 30 September 2010 (2010-09-30), pages 705 - 711 * |
| PANAGIOTIS ET AL: "PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method", 《JOURNAL OF MOLECULAR DIAGNOSTICS》, vol. 12, no. 5, 30 September 2010 (2010-09-30), pages 697 - 704, XP002685663, DOI: doi:10.2353/jmoldx.2010.100008 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816839A (en) * | 2012-07-06 | 2012-12-12 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene |
| CN102851368A (en) * | 2012-10-18 | 2013-01-02 | 广州达健生物科技有限公司 | PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method |
| CN103013993A (en) * | 2012-12-26 | 2013-04-03 | 武汉康圣达医学检验所有限公司 | Primer and method for mass spectrometric detection of hotspot mutation of PIK3CA genes by using primer |
| CN103013993B (en) * | 2012-12-26 | 2016-06-29 | 武汉康圣达医学检验所有限公司 | The method of primer and this primer Mass Spectrometer Method PIK3CA hotspot mutation |
| JP2017523810A (en) * | 2014-08-07 | 2017-08-24 | ファーマシスト リミテッドＰｈａｒｍａｓｓｉｓｔ Ｌｔｄ | Method for determining PIK3CA mutation status in a sample |
| US11279979B2 (en) | 2014-08-07 | 2022-03-22 | Pharmassist Ltd | Method of determining PIK3CA mutational status in a sample |
| CN104593515A (en) * | 2015-02-06 | 2015-05-06 | 中国医科大学 | PCR-RFLP method for detecting H-site mutation of PIK3CA gene |
| CN105969875B (en) * | 2016-06-15 | 2019-11-08 | 昆明理工大学 | Primer for detecting PIK3CA gene mutation in microcomponent combines and its application |
| CN105969875A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination |
| CN107574244A (en) * | 2016-07-05 | 2018-01-12 | 汪建平 | Detect primer pair, kit and the method for No. 1047 codon mutations of exon of PIK3CA genes 20 |
| CN107254529A (en) * | 2017-06-27 | 2017-10-17 | 郴州市第人民医院 | The kit and method of PIK3CA gene mutations in a kind of detection plasma DNA |
| CN107893109A (en) * | 2017-11-08 | 2018-04-10 | 重庆邮电大学 | A kind of low abundance gene mutation enrichment method based on removal wild-type sequence |
| CN107893109B (en) * | 2017-11-08 | 2021-07-06 | 重庆邮电大学 | Low-abundance gene mutation enrichment method based on removal of wild type sequence |
| CN108004317A (en) * | 2017-11-09 | 2018-05-08 | 上海赛安生物医药科技股份有限公司 | PIK3CA detection in gene mutation system and its kit |
| CN111363827A (en) * | 2020-04-30 | 2020-07-03 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting PIK3CA gene mutation and application method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102533958A (en) | Method and kit for detecting human PIK3CA gene mutation | |
| CN101978076B (en) | The method of the IGF1R/Chr15 in use FISH technology for detection circulating tumor cell | |
| CN102586401A (en) | Method and kit for detecting mutation of BRAF gene of human colorectal cancer | |
| WO2018086263A1 (en) | Real-time fluorescent quantitative pcr detection method, and standard sample and detection kit thereof | |
| Polivka Jr et al. | Testing for oncogenic molecular aberrations in cell-free DNA-based liquid biopsies in the clinic: are we there yet? | |
| Setty et al. | A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples | |
| JP2009511008A (en) | Method for predicting or monitoring a patient's response to an ErbB receptor drug | |
| CN101092644B (en) | Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung | |
| Wang et al. | Mutation detection and molecular targeted tumor therapies | |
| CN102115792A (en) | Method and kit for detecting KRAS gene mutations in human colon and rectum cancers | |
| CN105803088B (en) | The primer and probe and its kit of one group of detection Kras gene mutation | |
| CN103382503B (en) | EGFR gene 19 exon 19 kinds of deletion mutantion detection kit and detection method | |
| CN105256021B (en) | The method and its kit of Sensitive Detection human EGFR gene mutations are sequenced based on Sanger | |
| Marchoudi et al. | Distribution of KRAS and BRAF mutations in Moroccan patients with advanced colorectal cancer | |
| CN104017887A (en) | Primer pair as well as probe and kit for detecting human BRAF gene mutation | |
| CN102808027A (en) | EGFR (epidermal growth factor receptor) gene mutation site detection kit | |
| CN105441533A (en) | PIK3CA gene mutation detection system and kit thereof | |
| US20150344964A1 (en) | Prediction of the treatment response to an anti-egfr molecule in colorectal cancer patients | |
| WO2012131092A2 (en) | Method and kits for the prediction of response/nonresponse to the treatment with an anti-egfr antibody in patients with colorectal cancer of all uicc stages | |
| US20180112271A1 (en) | Kit For Detecting PIK3CA Gene Mutation | |
| EP2956554B1 (en) | A novel egfr variant | |
| CN108004320A (en) | EGFR genetic mutation detection architecture and its kit | |
| CN108728538B (en) | ALK gene fusion detection primer, method and kit | |
| Zaman et al. | Aberrant epidermal growth factor receptor RNA splice products are among the most frequent somatic alterations in clear cell renal cell carcinoma and are associated with a poor response to immunotherapy | |
| Wistuba | Molecular testing of non–small cell lung carcinoma biopsy and cytology specimens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120704 |