Detection of mutations by fill-in ligation reaction with enzyme-linked immunosorbent assay for rapid medical diagnosis

Abstract

Several approaches for parallel genotyping have been developed with increasingly available information on DNA variation. However, these methods require either complex laboratory procedures or expensive instrumentation. None of these procedures is readily performed in local clinical laboratories. In this study, we developed a flexible genotyping method involving fill-in ligation reaction with enzyme-linked immunosorbent assay successfully applied to detect important single-nucleotide polymorphisms (SNPs) for EGFR c.2573T > G (L858R), EGFR c.2582T > A (L861Q), and EGFR c.2155G > T (G719C). This assay exhibited excellent specificity, with a sensitivity as low as 0.5%. Eight out of 62 clinical samples were identified as heterozygotes for the SNP site of L858R, whereas only two samples were identified as heterozygotes by direct sequencing. The developed method enabled accurate identification of SNP in a simple and cost-effective manner adapted to routine analysis.

Graphical Abstract

The developed method was performed by four steps for mutation detection: FL reaction, purification, amplification and labeling, and ELISA detection.

Key words:

• genotyping
• mutations
• SNP
• fill-in ligation reaction
• EGFR

Funding

This research was supported by the research project of the Education Department of Hubei Province of China [grant number Q20132604]; the Science and Technology Project of Xiangyang City [grant number 201243]; and the Hubei University of Arts and Science [grant number 2012ya006].