Key result
Single-tube nested PCR with long internally quenched probes improves norovirus genogroup II detection sensitivity.
Why the study?
Does the use of long internally quenched probes in single-tube nested real-time PCR improve the sensitivity of detecting norovirus genogroup II?
Does the use of long internally quenched probes in single-tube nested real-time PCR improve the sensitivity of detecting norovirus genogroup II?
The use of long internally quenched probes in single-tube nested real-time PCR improves the analytical sensitivity for detecting highly variable RNA viruses like norovirus genogroup II.
May advance lab detection of variable RNA viruses; leaves open clinical diagnostic validation.
The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
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Xia et al. (2016) studied Norovirus genogroup II. Single-tube nested real-time PCR assays with long internally quenched probes vs. Traditional quenchers was evaluated on Assay sensitivity. Single-tube nested real-time PCR assays using long primers and probes with internal quenchers improved sensitivity for the detection of norovirus genogroup II.
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