A Viability Quantitative PCR Dilemma: Are Longer Amplicons Better?
Wannes Van Holm, Justien Ghesquière, Nico Boon, Tim Verspecht, Kristel Bernaerts, Naiera Zayed, Ioanna Chatzigiannidou, Wim Teughels
Abstract
Viability quantitative PCR (v-qPCR) has evolved into a valuable, mainstream technique for determining the number of viable microorganisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently, there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live/dead distinction.