We previously described the quick multiplex consensus PCR (QMC-PCR) as a method for rapid mutation screening in low-quality template. QMC-PCR has two-stages: a prediagnostic multiplex (PDM) reaction followed by a single specific diagnostic reaction with high-resolution melting (HRM) analysis. We aimed to develop QMC-PCRx in which second stage was multiplexed to allow testing of multiple targets.Methods
The PDM reaction was retained without change. For the second stage, in silico design was used to identify targets amenable to a multiplex specific diagnostic reaction and multiplex HRM (mHRM) analysis. Following optimisation, 17 colorectal cancers were tested for mutation in five hotspots. For QMC-PCR, each target was tested individually. For QMC-PCRx, the targets were tested in the following combinations (i) KRAS exon 3/PIK3CA exon 20/PTEN exon 3 in triplex and (ii) PTEN exon 7/NRAS exon 2 in duplex. The degree of agreement between the novel QMC-PCRx and the standard QMC-PCR was tested by the percentage concordance.Results
Optimisation of mHRM showed that peaks needed to be separated (without overlap) and the optimal number was three targets per test. Our experimental design produced distinct and widely separated peaks for the individual targets although one of the primers needed a GC-tail. A total of 85 individual targets were tested; this required 85 second-stage PCR/HRM tests by QMC-PCR versus 34 second-stage tests by QMC-PCRx. The percentage concordance between the singleplex and multiplex methodologies was 100%.Conclusions
A multiplexed analysis using HRM is possible without loss of diagnostic accuracy. The novel QMC-PCRx protocol can significantly reduce workload and costs of mutation screening.