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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue

Guoqiang Li1 / Xiaodi Luo1 / Jiaying He1 / Zeyao Zhu1 / Gang Yu1 / Huijuan Qin1 / Tao Zeng1 / Zhiming Liu1 / Shiyang Wu1 / Jiasen Xu1 / 1

1SurExam Bio-Tech Co. Ltd., Guangzhou Technology Innovation Base, Science City, Guangzhou, P.R. China

Corresponding author: Lifen Ren-Heidenreich, Guangzhou SurExam Bio-Tech Co. Ltd., Guangzhou, Guangdong, P.R. China

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 49, Issue 2, Pages 191–195, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2011.040, December 2010

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Background: DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex®-xTAG70plex-EGFR liquidchip.

Methods: This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized.

Results: A total of 70 alleles of somatic mutations of EGFR, KRAS, BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was <8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%–5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected.

Conclusions: A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR, KRAS, BRAF and PIK3CA gene mutations from FFPE tumor slides.

Keywords: allele specific primer extension; EGFR signaling pathway; liquidchip; somatic mutations

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