Method for detecting EGFR G719X gene mutation by using digital PCR (polymerase chain reaction) technology
A technology for gene mutation and technical detection, applied in the field of detecting EGFRG719X gene mutation using digital PCR technology, can solve the problem of low sensitivity of EGFRG719X gene mutation detection, avoid false negative results, reduce clinical testing cost and workload, and design specificity strong effect
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Embodiment 1
[0031] Example 1: Design and synthesis of primers and probes for detection of EGFR G719X gene variation by ddPCR
[0032] For the three variant forms of EGFR G719X of exon 18 of the EGFR gene, using the full-length cDNA of exon 18 of the EGFR gene as a template, use Oligo software to analyze the sites of TaqMan primers and probes, and select the best combination as follows:
[0033] G719X variant upstream and downstream primers:
[0034]Upstream primer SEQ NO1: 5'-GCTCCCAACCAAGCTCTCT-3'
[0035] Downstream primer SEQ NO2: 5'-CCTTATACACCGTGCCGAAC-3'
[0036] When using the above primers for PCR amplification, the amplified fragment is 94bp, which is especially suitable for the amplification of small fragment DNA samples such as plasma free DNA.
[0037] The designed G719X mutation detection probes are all fluorescently labeled, with a reporter group at the 5' end and a non-fluorescent quencher group at the 3' end. The sequence is as follows:
[0038] G719A detection probe is...
Embodiment 2
[0043] Example 2: Detection of EGFR G719X Gene Variation in Whole Blood Samples
[0044] 1. Prepare the samples to be tested: DNA containing wild-type EGFR gene, three variant forms of EGFR exon 18 (G719A, G719S, G719C) mutation-positive DNA, and wild-type mutant DNA mixed samples (wherein the mutant and wild-type The content ratios are 1 / 100, 1 / 1000, 1 / 10000, respectively). DNA is derived from plasma. Wherein, the mutant DNA template is derived from G719A, G719S, G719C gene variant cell lines carrying EGFR exon 18 (identified by PCR sequencing).
[0045] 2. Extraction of cfDNA: Use a kit to extract cfDNA. For specific operations, refer to the instructions of the QIA ampDNA Mini Kit kit from QIAGEN.
[0046] 3. Prepare the PCR reaction solution in the PCR plate according to the following ratio: 2× digital PCR master mix (Biorad, #1863010), upstream and downstream primers for detecting EGFR G719X gene variation, detection probe for EGFR G719X gene variation and The cfDNA tem...
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