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. 2025 Jan;17(1):49-62.
doi: 10.1080/17576180.2025.2451527. Epub 2025 Jan 15.

Development and validation of an ultrasensitive qPCR method to identify and quantify EGFR T790M in cell-free DNA

Shenglei Yuan  1 , Nan Jia  1 , Guofu Lu  1 , Jinping Lai  2 , Wenzhong Liang  1 , Lan Li  1 , Chenpu Zhang  1 , Jianbo Diao  1
Affiliations

Development and validation of an ultrasensitive qPCR method to identify and quantify EGFR T790M in cell-free DNA

Shenglei Yuan et al. Bioanalysis. 2025 Jan.

Abstract

Background: Circulating tumor DNA (ctDNA) is a promising biomarker for cancer prognosis and drug development. A major challenge in the ctDNA determination method is discriminating ctDNA from highly similar but significantly more abundant wild-type DNA sensitively and accurately.

Method: An ultrasensitive qPCR method termed Triple Enrichment Amplification of Mutation PCR (TEAM-PCR) was developed to detect EGFR T790M mutation.

Results: EGFR T790M was quantified over the assay range of 25-106 copies/reaction in the presence of 106 wild-type copies. This method was fully validated following the essential bioanalysis guidance, with the limit of detection (LOD) being five copies/reaction.

Conclusion: This study established and validated a qPCR-based strategy to detect EGFR T790M mutation with ultra-high sensitivity and reliability.

Keywords: EGFR T790M; LOD; NSCLC; TEAM-PCR; bioanalysis; ctDNA; method validation; point mutation; qPCR; sensitivity.

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Conflict of interest statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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