Method for detecting EGFR G719X gene mutation by using digital PCR (polymerase chain reaction) technology
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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
Inactive Publication Date: 2018-04-20
PRIMBIO GENES BIOTECH WUHAN CO LTD
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[0005] In order to overcome the above-mentioned deficiencies of the prior art, the object of the present invention is to propose a method for detecting EGFR G719X gene variation using digital PCR technology for the defect of low detection sensitivity of EGFRG719X gene variation in non-tumor tissues of patients
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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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Abstract
The invention discloses a method for detecting EGFR G719X gene mutation by using a digital PCR (polymerase chain reaction) technology. The method comprises the steps of taking the digital PCR as a detection platform; designing upstream and downstream primes and probes, used for detecting the EGFR G719X gene mutation, by aiming to three deviant forms of an EGFR G719X gene; mixing a cfDNA template of a to-be-detected sample, the upstream and downstream primes and probes used for detecting the EGFR G719X gene mutation and a PCR pre-mixed solution to prepare ddPCR micro-reaction drops to perform PCR amplification reaction; judging whether an EGFR G719X gene mutation template is contained in the to-be-detected sample or not and calculating the content of the mutated template. The primes and theprobes provided by the invention are high in specificity, when being applied to the PCR detection, the sensitivity can reach 0.01%; in addition, by adopting the set of primers to carry out the PCR amplification, an amplified fragment is only 94bp, is especially suitable for the amplification of small fragment DNA samples such as plasma free DNA, the mutation with extremely low abundance can be detected out from the cfDNA, and an important role on guiding clinic treatment and improving patient prognosis is realized; the method can be used for detecting the three deviant forms of the EGFR G719Xgene at one time, so that the clinical test cost and workload are greatly reduced.
Description
technical field [0001] The invention relates to molecular biological detection of genes in the field of biotechnology, in particular to a method for detecting EGFR G719X gene variation by using digital PCR technology. Background technique [0002] Non-small cell lung cancer (NSCLC) is a malignant tumor that seriously threatens human health. Despite the continuous improvement of surgery and chemotherapy techniques, the prognosis of patients is still poor, and the 5-year survival rate is less than 20%. At present, molecular targeted therapy targeting human epidermal growth factor receptor (EGFR) has become the most important way to treat NSCLC. [0003] The currently clinically used targeted drug against EGFR is EGFR tyrosine kinase inhibitor (EGFR-TKI). Targeted therapy. The efficacy of EGFR-TKI is closely related to the mutation status of EGFR gene. The mutations in the EGFR tyrosine kinase region mainly occur in exons 18-21, and the mutations in exons 19 and 21 cover 90% ...
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