CN105713965A - Kit for detecting PIK3CA gene mutation - Google Patents
Kit for detecting PIK3CA gene mutation Download PDFInfo
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- CN105713965A CN105713965A CN201510836702.8A CN201510836702A CN105713965A CN 105713965 A CN105713965 A CN 105713965A CN 201510836702 A CN201510836702 A CN 201510836702A CN 105713965 A CN105713965 A CN 105713965A
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Abstract
The invention discloses a kit for detecting PIK3CA gene mutation, which comprises peptide nucleic acids and specific primers with the following sequences: (1) peptide nucleic acid and the specific primer aiming at the ninth exon of a PIK3CA gene: shown as SEQ ID NO.1-3; and (2) peptide nucleic acid and the specific primer aiming at the twentieth exon of a PIK3CA gene: shown as SEQ ID NO.4-6. A method for detecting the PIK3CA gene mutation by utilizing the kit comprises the following steps: (1) extracting the DNA of a to-be-detected sample; (2) by taking the extracted DNA as a template, carrying out quantitative PCR amplification by utilizing the kit for detecting the PIK3CA gene mutation, and detecting the value of deltaCt; and (3) making judgment, if deltaCt is greater than 8, judging that the genotype of the PIK3CA gene of the to-be-detected sample is the wild type, and if deltaCt is smaller than 8, judging that the genotype of the PIK3CA gene of the to-be-detected sample is the mutation type. The kit has the advantages that the operation is easy and convenient, the time consumed is short, the sensitivity is high, the detection result is accurate and reliable, etc.
Description
Technical field
The present invention relates to a kind of test kit detecting PIK3CA gene mutation.
Background technology
PIK3CA gene was utilized hybridization in situ technique to detect by Volinia in 1994, the p110 catalytic subunit of coding I class phosphatidylinositol-3-kinase (phosphatidylino-sitol3-kinases, PI3Ks).The sudden change of the 9th exon and the 20th exon is possible not only to reduce the apoptosis of cell and can also promote the infiltration of tumor, improve the activity of kinases PI3Ks downstream.Its sudden change improves the activity of kinases PI3Ks downstream, it is possible to reduce the apoptosis of cell can also promote the infiltration of tumor.At present, detection method clinically needs first to carry out gene fragment amplification, then utilizes the method detection gene mutation of order-checking.The method is relatively costly, and testing cost is expensive.Being additionally, since in gene fragment amplification process, mutant gene and wild gene have all been carried out the amplification of equal extent, therefore when order-checking detection, sensitivity is not high, only has about 10%, and operates comparatively laborious, consuming time longer.Additionally, for blood preparation, owing to sensitivity is low, so being difficult to sudden change therefrom be detected.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of test kit detecting PIK3CA gene mutation, it has highly sensitive, the testing result advantage such as accurately and reliably.
The present invention is achieved by the following technical solutions:
A kind of test kit detecting PIK3CA gene mutation, including following sequence of peptide nucleic acid(PNA) and specific primer:
(1) for the peptide nucleic acid(PNA) of PIK3CA gene the 9th exon and specific primer:
The sequence of described peptide nucleic acid(PNA) is: 5 '-TCTGAAATCACTGAGCAG-3 '；As shown in SEQIDNO.1；
The sequence of described specific primer is:
Forward primer: 5 '-GAGACAATGAATTAAGGGAAAATGA-3 '；As shown in SEQIDNO.2；
Reverse primer: 5 '-CTCCATAGAAAATCTTTCTCCTGCT-3 '；As shown in SEQIDNO.3；
(2) for the peptide nucleic acid(PNA) of PIK3CA gene the 20th exon and specific primer:
The sequence of described peptide nucleic acid(PNA) is: 5 '-TGATGCACATCATGGTG-3 '；As shown in SEQIDNO.4；
The sequence of described specific primer is:
Forward primer: 5 '-TGCATACATTCGAAAGACCCTAG-3 '；As shown in SEQIDNO.5；
Reverse primer: 5 '-AATCCATTTTTGTTGTCCAGCCA-3 '；As shown in SEQIDNO.6；.
Further, described test kit also includes the necessary reaction raw materials of PCR reaction and reagent.
The necessary reaction raw materials of described PCR reaction and reagent include: PCR buffer, Mg2+With the reactant mixture of dNTPs, Taq enzyme, SuperGreen dyestuff, ultra-pure water.
A kind of method utilizing the detection PIK3CA gene mutation of mentioned reagent box, comprises the following steps:
(1) DNA (DNA extraction kit description conventionally is operated) of sample to be detected (including various clinical samples, be particularly suited for blood preparation) is extracted；
(2) with the DNA of said extracted for template, utilize the test kit of above-mentioned detection PIK3CA gene mutation to carry out quantitative pcr amplification, detect Δ Ct value (Δ Ct=Sample+PNA-Sample-PNA), (quality-control product must add, and positive quality control product is confirm the cell strain DNA of PIK3CA positive gene mutation through order-checking to set up Quality Control comparison；Negative quality-control product is confirm, through order-checking, the cell strain DNA that PIK3CA gene mutation is negative)；
Described pcr amplification program is: 95 DEG C 5 minutes；95 DEG C 10 seconds, 70 DEG C 20 seconds, 58 DEG C 20 seconds, 70 DEG C 20 seconds, repeat 45 circulations；
(3) judge: if Δ Ct > 8, then the genotype of the PIK3CA gene of sample to be detected is wild type；If Δ Ct < 8, then the genotype of the PIK3CA gene of sample to be detected is saltant type.
The test kit of the present invention is that the characteristics design utilizing peptide nucleic acid(PNA) (peptidenucleicacids, PNA) forms.PNA is the nucleic acid analog of a kind of synthetic, and it is in combinations with unique DNA template, and cannot function as the synthesis of primer inducing DNA.Compared with common nucleic acid, annealing temperature exceeds 50%.The PNA sequence of our design and wild type gene complete complementary, so, when PNA and DNA masterplate has a base not mate (template exists sudden change), adhesion between them will fall rapidly upon, gene with sudden change is expanded thus being enriched with, and the amplification of wild gene is suppressed.Finally, evaluating testing result according to Δ Ct value, specific standards is: during Δ Ct > 8, and specimen is wild type；During Δ Ct < 8, specimen is PIK3CA genic mutation type (Δ Ct=Sample+PNA-Sample-PNA)。
The present invention is for detecting test kit and the detection method of PIK3CA gene mutation, and easy and simple to handle, consuming time short, highly sensitive, testing result accurately and reliably, has the advantage that
1. this test kit is less costly, reduces the testing cost of gene mutation.
2., in detection process, by the amplification to the suppression of wild type and saltant type, it is finally reached the purpose of enrichment mutated genes fragment, is greatly promoted the sensitivity of sudden change detection.
3., owing to sensitivity is high, so blood sample gene mutation recall rate is fine, the patient for being difficult to obtain tissue specimen clinically provides detection new way.
4. result presents simply, easy to operate.
5. can be used for extensive tumor high-risk examination, the early diagnosis of tumor and the dynamic of curative effect prognosis to observe.
Accompanying drawing explanation
Fig. 1: mutant gene accounts for template total amount ratio when being 50%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 2: mutant gene accounts for template total amount ratio when being 25%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 3: mutant gene accounts for template total amount ratio when being 12.5%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 4: mutant gene accounts for template total amount ratio when being 6.25%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 5: mutant gene accounts for template total amount ratio when being 3.1%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 6: mutant gene accounts for template total amount ratio when being 1.5%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 7: mutant gene accounts for template total amount ratio when being 0.75%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 8: mutant gene accounts for template total amount ratio when being 0.38%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Fig. 9: mutant gene accounts for template total amount ratio when being 0.19%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Figure 10: mutant gene accounts for template total amount ratio when being 0%, adds PNA and is added without the amplified fluorescence curve (adding the amplification curve of PNA front) of PNA.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Instrument involved in following embodiment, reagent, material etc., unless otherwise noted, be in prior art existing conventional instrument, reagent, material etc., can pass through regular being either commercially available.Experimental technique involved in following embodiment, detection method etc., unless otherwise noted, it is in prior art existing normal experiment method, detection method etc..
Embodiment 1
A kind of test kit detecting PIK3CA gene mutation
Utilizing mentioned reagent box to detect, step is as follows:
(1) cell line (the 9th exon c.1633G > A of PIK3CA gene masculine is cultivated；P.E545K mutational cell line: DLD-1；20th exon c.3140A > G；P.H1047R mutational cell line: HCT116) and negative cells system A549[all buy from American Type Culture institute (AmericanTypeCultureCollection) (Manassas, VA, USA)].Extract its DNA, quantitatively.Ultimate density is diluted to 25ng/ul.
(2) negative cells system DNA is mixed in positive cell line DNA by different proportion, finally prepare the sample (50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%, 0.38%, 0.19%, 0%) that positive DNA ratio is different.
(3) DNA taking 2ul difference positive rate respectively is made directly the quantitative pcr amplification adding with being not added with PNA, and PCR system is 20ul altogether, including 2ul10 × PNA；The each 0.5ul of forward and reverse primer；1ulTaq enzyme；1ulSuperGreen dyestuff；10ulPCR reactant liquor；4ulH2O or 6ulH2O (is not added with PNA).Use ROCHELightCycler480 quantitative PCR apparatus.95 DEG C 5 minutes；95 DEG C 10 seconds, 70 DEG C 20 seconds, 58 DEG C 20 seconds, 72 DEG C 20 seconds, repeat 45 circulations.
Result is Fig. 1～10 such as, and shown in table 1.If during with Δ Ct > 8, specimen is wild type；During Δ Ct < 8, the standard that specimen is PIK3CA genic mutation type judges, then the detection sensitivity of this test kit is 1%.
Table 1
*:Δ ct=Sample+PNA-Sample-PNAThis table is the statistical table after being calculated by the amplification curve of Fig. 1～10.
Along with in template DNA, mutant DNA ratio reduces, wild type DNA ratio improves accordingly, then the effect of PNA is further notable, is embodied in Δ ct value and is gradually increased.
The specific embodiment of the present invention is described although above-mentioned in conjunction with the embodiments; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme, those skilled in the art need not pay various amendments or deformation that creative work can make still within protection scope of the present invention.
Claims (5)
1. the test kit detecting PIK3CA gene mutation, it is characterised in that: include following sequence of peptide nucleic acid(PNA) and specific primer:
(1) for the peptide nucleic acid(PNA) of PIK3CA gene the 9th exon and specific primer:
The sequence of described peptide nucleic acid(PNA) is: 5 '-TCTGAAATCACTGAGCAG-3 '；
The sequence of described specific primer is:
Forward primer: 5 '-GAGACAATGAATTAAGGGAAAATGA-3 '；
Reverse primer: 5 '-CTCCATAGAAAATCTTTCTCCTGCT-3 '；
(2) for the peptide nucleic acid(PNA) of PIK3CA gene the 20th exon and specific primer:
The sequence of described peptide nucleic acid(PNA) is: 5 '-TGATGCACATCATGGTG-3 '；
The sequence of described specific primer is:
Forward primer: 5 '-TGCATACATTCGAAAGACCCTAG-3 '；
Reverse primer: 5 '-AATCCATTTTTGTTGTCCAGCCA-3 '.
2. the test kit of detection PIK3CA gene mutation according to claim 1, it is characterised in that: described test kit also includes the necessary reaction raw materials of PCR reaction and reagent.
3. the test kit of detection PIK3CA gene mutation according to claim 2, it is characterised in that: the necessary reaction raw materials of described PCR reaction and reagent include: PCR buffer, Mg2+With the reactant mixture of dNTPs, Taq enzyme, SuperGreen dyestuff, ultra-pure water.
4. the method for the test kit detection PIK3CA gene mutation of the detection PIK3CA gene mutation that a kind utilizes according to any one of claim 1～3, it is characterised in that: comprise the following steps:
(1) DNA of sample to be detected is extracted；
(2) with the DNA of said extracted for template, utilize the test kit of detection PIK3CA gene mutation to carry out quantitative pcr amplification, detect Δ Ct value, set up Quality Control to compare；
(3) judge: if Δ Ct > 8, then the genotype of the PIK3CA gene of sample to be detected is wild type；If Δ Ct < 8, then the genotype of the PIK3CA gene of sample to be detected is saltant type.
5. method according to claim 4, it is characterised in that: described pcr amplification program is: 95 DEG C 5 minutes；95 DEG C 10 seconds, 70 DEG C 20 seconds, 58 DEG C 20 seconds, 70 DEG C 20 seconds, repeat 45 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510836702.8A CN105713965A (en) | 2015-11-23 | 2015-11-23 | Kit for detecting PIK3CA gene mutation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593856A (en) * | 2019-01-24 | 2019-04-09 | 宋现让 | A kind of kit and preparation method thereof of detection 2 codon 12/13 of KRAS gene extron mutation |
| 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 (3)
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| WO2012020965A2 (en) * | 2010-08-13 | 2012-02-16 | 주식회사 파나진 | Pik3ca mutation detection method and kit using real-time pcr clamping of pna |
| CN104099422A (en) * | 2014-07-18 | 2014-10-15 | 普世华康江苏医疗技术有限公司 | Composition for detecting colorectal cancer hotspot gene mutation sites and using method of composition |
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2015
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Patent Citations (3)
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| WO2012020965A2 (en) * | 2010-08-13 | 2012-02-16 | 주식회사 파나진 | Pik3ca mutation detection method and kit using real-time pcr clamping of pna |
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Non-Patent Citations (3)
| Title |
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| AHRIM MOON等: "EGFR, COX2, p-AKT expression and PIK3CA mutation in distal extrahepatic bile duct carcinoma", 《PATHOLOGY》 * |
| MI JUNG KWON等: "Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue", 《PATHOLOGY – RESEARCH AND PRACTICE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593856A (en) * | 2019-01-24 | 2019-04-09 | 宋现让 | A kind of kit and preparation method thereof of detection 2 codon 12/13 of KRAS gene extron mutation |
| 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 |
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