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Digital PCR detection method for human PIK3CA gene mutation and application

A technology of use and reaction system, applied in the field of digital PCR detection of human PIK3CA gene mutation, can solve the problems of unsatisfactory sensitivity and specificity

Pending Publication Date: 2022-01-14
铭炽生物科技(上海)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Douillard et al. reported that detection of EGFR mutations using plasma concordance with tumor tissue detection was only 65%
[0008] In addition, although there are some methods based on cfDNA to detect PIK3CA gene mutations, the sensitivity and specificity of these methods are not satisfactory.

Method used

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  • Digital PCR detection method for human PIK3CA gene mutation and application
  • Digital PCR detection method for human PIK3CA gene mutation and application
  • Digital PCR detection method for human PIK3CA gene mutation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] The screening of embodiment 1 primer pair

[0209] According to the sequence of the 9th exon of PIK3CA (E545, E542 sites), design 5 pairs of primers (primers were synthesized from Shanghai Sangon Biotechnology Co., Ltd.), the specific sequences are shown in Table 3.

[0210] Table 3. Screening 5 pairs of primers for amplification of PIK3CA E545, E542

[0211]

[0212]

[0213] According to the sequence of exon 20 of PIK3CA (H1047 site), 5 pairs of primers were designed (primers were synthesized from Shanghai Sangon Biotechnology Co., Ltd.), and the specific sequences are shown in Table 4.

[0214] Table 4. Screening of 5 pairs of primers used to amplify PIK3CA H1047

[0215]

[0216] Screening PCR system: 0.2ul of Taq HS polymerase, 2ul of 10×HS PCR buffer, 1.6ul of dNTP mixture, 1ul of upstream primer, 1ul of downstream primer, 5ul of tgDNA (human leukocyte genomic DNA, as a template), and make up the system with water to 20ul .

[0217] Screening PCR progr...

Embodiment 2

[0219] Embodiment 2 Taqman probe and the optimization of digital PCR reaction program

[0220] In this example, Taqman probes were synthesized and optimized, and the digital PCR reaction program was optimized based on the optimized Taqman probes.

[0221] 2.1 Taqman probe

[0222] In this embodiment, the detected PIK3CA gene mutations are nucleotide mutations corresponding to E545K, E542K, and H1047R (Table 5);

[0223] Table 5: PIK3CA gene mutation information table

[0224] Detection site mutation type COSMIC number genetic change E545K point mutation COSM763 c.1633G>A E542K point mutation COSM760 c.1624G>A H1047R point mutation COSM775 c.3140A>G

[0225] Based on the genome sequence and mutation site of human PIK3CA, the corresponding Taqman probes were designed and optimized. The optimized Taqman probes contained specific locked nucleotides (+N), respectively WTP-E545 / MTP-E545K, WTP -E542 / MTP-E542K, WTP-H1047 / MTP-H104...

Embodiment 3

[0237] Example 3 Sensitivity Verification of Detection of PIK3CA Gene Mutation by Digital PCR

[0238] Calculate wild-type template copy number (copies / ul), mutant template genome copy number (copies / ul) and mutation rate (%) according to Example 1, and dilute both to 4000 copies / ul with TE. The tgDNA was mixed into the genome template containing the mutation, so that the ratio of the mutation template was 0.06%, 0.13%, 0.25%, 0.5%, and 1%, respectively, and a gradient dilution template was prepared.

[0239] Validate digital PCR system: 2×ddPCR Supermix (No dUTP) 11ul, primer 1 (E545 / E542-F, H1047-F) 1.1ul, primer 2 (E545 / E542-R, H1047-R) 1.1ul, probe 1 (WTP-E545, E542 / WTP-H1047) 0.55ul, probe 2 (MTP-E545K, MTP-E545K, MTP-H1047R) 0.55ul, template 5.5ul, add water to make up to 22ul.

[0240] Add templates in the sample preparation area in the following order: blank control, negative control, and serially diluted template. The blank control is water, the negative control is ...

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Abstract

The invention provides a digital PCR detection method for human PIK3CA gene mutation and application. Specifically, the invention provides a preferred primer pair aiming at sequences of PIK3CA E545, E542 and PIK3CA H1047, an amplification method, a nucleic acid probe and a detection system, and further provides a kit for detecting PIK3CA gene mutation. By optimizing the primer pair, the probe and corresponding reaction conditions, the PIK3CA gene mutation can be detected in a high-sensitivity and strong-specificity manner for different samples.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a digital PCR detection method and application of human PIK3CA gene mutation. Background technique [0002] PIK3CA is a proto-oncogene located on chromosome 3, with a total of 20 exons. About 80% of PIK3CA mutations occur in the two hot spots of the helical region and the kinase region. The three most common mutations are H1047R on exon 20, and E542K and E545K on exon 9. The mutation of PIK3CA gene can not only increase the catalytic activity of PI3Ks, but also promote cell canceration. [0003] PI3Ks are activated as downstream signaling molecules of EGFR, which can lead to the resistance of tumor cells to EGFR-TKI drugs. For example, the mutation of PIK3CA gene can lead to the resistance of cetuximab and panitumumab to the treatment of metastatic colorectal cancer, Lead to resistance of gefitinib and erlotinib to the treatment of non-small cell lung cancer. PIK3CA mutations are...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2563/159C12Q2563/107C12Q2561/101
Inventor 江萤童慧娟黄昕
Owner 铭炽生物科技(上海)有限公司
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