Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II

Hongyan Xia, Sabine Gravelsina, Christina Öhrmalm, Jakob Ottoson, Jonas Blomberg

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)28-34
Number of pages7
JournalBioTechniques
Volume60
Issue number1
DOIs
Publication statusPublished - Jan 2016

Keywords*

  • Norovirus
  • Real-time PCR
  • Variation-tolerant capture multiplex assay

Field of Science*

  • 1.6 Biological sciences

Publication Type*

  • 1.1. Scientific article indexed in Web of Science and/or Scopus database

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