A mismatch-tolerant RT-quantitative PCR: application to broad-spectrum detection of respiratory syncytial virus
Open Access
- 1 May 2019
- journal article
- research article
- Published by Future Science Ltd in BioTechniques
- Vol. 66 (5), 225-230
- https://doi.org/10.2144/btn-2018-0184
Abstract
Quantitative PCR (qPCR) is widely used to detect viruses. However, mismatches occurring in the 3′-end of the primers reduce amplification efficiency of qPCR and limit its capacity in detection of highly variable viruses. Here, we reported a mismatch-tolerant RT-qPCR with a small amount of additional high-fidelity DNA polymerase for simultaneous detection of RSV-A and RSV-B. The novel assay had higher amplification efficiency for various variants forming mismatches with the primers than the conventional RT-qPCR, and showed good specificity and sensitivity. It demonstrated a good correlation coefficient with a commercial RSV detection kit and had relatively lower Ct values than the kit for 16 of 20 RSV-positive samples. The mismatch-tolerant qPCR technique is a promising approach for sensitive detection of highly variable viruses. METHOD SUMMARY We developed a mismatch-tolerant RT-quantitative PCR by simply adding a small amount of additional high-fidelity DNA polymerase to the standard reaction mixtures. The new method well tolerates mismatches between primers and templates and is especially suited to the detection of highly variable viruses.Keywords
This publication has 33 references indexed in Scilit:
- The WHO R&D Blueprint: 2018 review of emerging infectious diseases requiring urgent research and development effortsAntiviral Research, 2018
- Emerging infectious diseases in Africa in the 21st centuryNew Microbes and New Infections, 2018
- Quasispecies and virusEuropean Biophysics Journal, 2018
- Emerging infectious diseasesMedicine, 2014
- Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplificationBiomedical Microdevices, 2012
- What Does Virus Evolution Tell Us about Virus Origins?Journal of Virology, 2011
- Viral Mutation RatesJournal of Virology, 2010
- Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR methodJournal of Virological Methods, 2009
- The real-time polymerase chain reactionMolecular Aspects of Medicine, 2006
- The Distribution of Rates of Spontaneous Mutation over Viruses, Prokaryotes, and EukaryotesAnnals of the New York Academy of Sciences, 1999