Method and kit for non-invasively detecting EGFR gene mutations

A method for non-invasively detecting an EGFR gene mutation in a subject, comprising the following steps:

• designing a pair of forward and reverse primers that are adjacent and backward extended,
• targeting an exon of the EGFR gene wherein the exon is selected from the group consisting of exon 18, exon 19, exon 20, or exon 21, wherein the forward and reverse primer pairs that target exon 18 are selected from a group consisting of
• E18-1-3Forward:
• CCCAGCTTGTGGAGCCTC [SEQ ID NO.:1];
• E18-1-3Reverse:
• GACAAGAACACAGAGACAAGGGT [SEQ ID NO.:2] and
• E18-2-Forward:
• GCAGGGCCTCTCATGGTC [SEQ ID NO.:3];
• E18-2-Reverse:
• CCTGTGCCAGGGACCTTAC [SEQ ID NO.:4],
• wherein the forward and reverse primer pairs that target exon 19 are selected from a group consisting of
• E19-1-Forward:
• ACGTCTTCCTTCTCTCTCTGTCAT [SEQ ID NO.:5];
• E19-1-Reverse:
• GTGAGATGGTGCCACATGCT [SEQ ID NO.:6] and
• E19-2-Forward:
• GTCCATGGCTCTGAACCTCA [SEQ ID NO.:7];
• E19-2-Reverse:
• CCACACAGCAAAGCAGAAAC [SEQ ID NO.:8],
• wherein the forward and reverse primers that target exon 20 are
• E20-2-1 Forward:
• CCTCCCCGTATCTCCCT [SEQ ID NO.:11];
• E20-2-1 Reverse:
• GGAGATAAGGAGCCAGGAT [SEQ ID NO.:12], or
• wherein the forward and reverse primer pairs that target exon 21 are selected from a group consisting of
• E21-1-Forward:
• AGCAGGGTCTTCTCTGTTTCA [SEQ ID NO.:13];
• E21-1-Reverse:
• GAGGGACAGATCATCATGGG [SEQ ID NO.:14] and
• E21-2-Forward:
• TTTCCTGACACCAGGGACC [SEQ ID NO.:15];
• E21-2-Reverse:
• TGACCTAAAGCCACCTCCTT [SEQ ID NO.:16];
• extracting plasma DNAs from the subject;
• connecting the extracted plasma DNAs with tagging linkers;
• PCR pre-amplifying the tagging linkers connected plasma DNAs;
• cyclizing the pre-amplified DNAs to obtain cyclized DNAs, wherein the cyclization is a splint mediated single strand DNA cyclization;
• PCR amplifying the cyclized DNAs using the designed primers;
• and
• high throughput sequencing the PCR amplified product and analyzing the EGFR gene mutations.