Taqman-MGB nanoPCR for Highly Specific Detection of Single-Base Mutations.

Abstract

PurposeDetection of single-base mutations is important for real-time monitoring of tumor progression, therapeutic effects, and drug resistance. However, the specific detection of single-base mutations from excessive wild-type background sequences with routine PCR technology remains challenging. Our objective is to develop a simple and highly specific qPCR-based single-base mutation detection method.MethodsUsing EGRF T790M as a model, gold nanoparticles at different concentrations were separately added into the Taqman-MGB qPCR system to test specificity improvement, leading to the development of the optimal Taqman-MGB nanoPCR system. Then, these optimal conditions were used to test the range of improvement in the specificity of mutant-type and wild-type templates and the detection limit of mutation abundances in a spiked sample.ResultsThe Taqman-MGB nanoPCR was established based on the traditional qPCR, with significantly suppressed background noise and improved specificity for single-base mutation detection. With EGFR T790M as a template, we demonstrated that our Taqman-MGB nanoPCR system could improve specificity across a wide concentration range from 10−9 μM to 10 μM and detect as low as 0.95% mutation abundance in spiked samples, which is lower than what the traditional Taqman-MGB qPCR and existing PCR methods can detect. Moreover, we also proposed an experimentally validated barrier hypothesis for the mechanism of improved specificity.ConclusionThe developed Taqman-MGB nanoPCR system could be a powerful tool for clinical single-base mutation detection.