Real-time amplification and high resolution melt analysis on a rapid microfluidic instrument

Abstract

Background

Real-time nucleic acid amplification represents a robust, ubiquitous technology that often requires additional downstream analysis to identify sequence polymorphisms or differentiate specific from non-specific amplification (NSA). For example, common post-amplification analysis methods for the polymerase chain reaction (PCR) include sequencing and electrophoresis, both of which are labor- and time-intensive techniques that require expensive additional reagents and consumables. In contrast, high resolution melt (HRM) analysis presents a simpler alternative that can elucidate sequence differences and distinguish specific from non-specific amplification without requiring separate instrumentation or additional reagents beyond those used for real-time amplification.

Results

We have previously reported a microfluidic real-time amplification system that could complete PCR in 8 minutes with comparable sensitivity to conventional instruments that require 50+ minutes for the same assay. Here, we describe expanding the capability of the system to include post-amplification HRM analysis. Sequence differentiation was demonstrated using PCR to detect epigenetic targets with different methylation percentages. Moreover, isothermal amplification methods that commonly experience NSA due to excessive primer noise, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), were coupled with HRM to distinguish true positive results. All microfluidic experiments were completed in parallel on conventional benchtop instrumentation, the results of which were found to be comparable with the microfluidic system consuming a fraction of the total analysis time required on the conventional instrument.

Significance

With these modifications, the microfluidic platform, which has demonstrated 8 minute PCR, can perform HRM in under 4 minutes following amplification for differentiation of sequences containing mutations and elucidating NSA. We demonstrate enhanced applicability of this microfluidic instrument for point-of-need applications, including clinical diagnostics, where rapid and accurate genomic analysis is paramount.