Superiority of digital RT-PCR over real-time RT-PCR for quantitation of highly divergent human rhinoviruses.

Publication Type:

Journal Article

Source:

Journal of clinical microbiology (2016)

Abstract:

Human rhinoviruses (HRV) are comprised of 3 species, represented by over 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for the development of robust molecular methods that detect all genotypes with equal efficiency. This study compares the accuracy of reverse transcription-quantitative PCR (RT-qPCR) and reverse transcription-digital PCR (RT-dPCR) for quantification of HRV RNA using genotype-specific primers and probes and a consensus primer/probe set targeting the 5' non-coding region of HRV. When using consensus primers and probes for quantification of HRV, RT-dPCR outperformed RT-qPCR by consistently and accurately quantifying HRV RNA across more genotype groups, despite the presence of up to 2 target sequence mismatches within the primer or probe binding region. Because it does not rely on amplification efficiency, which can be affected by sequence mismatches in primer/probe binding regions, RT-dPCR may be the optimal molecular method for future HRV quantification studies, and for quantitation of other viruses with high sequence diversity.