Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

Helene Schulz; Mackenzie Neale; Vanessa Zubach; Alberto Severini; Joanne Hiebert

doi:10.1016/j.jviromet.2022.114500

Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

J Virol Methods. 2022 May:303:114500. doi: 10.1016/j.jviromet.2022.114500. Epub 2022 Feb 22.

Authors

Helene Schulz¹, Mackenzie Neale², Vanessa Zubach³, Alberto Severini⁴, Joanne Hiebert⁵

Affiliations

¹ Viral Exanthemata and STD Section, National Microbiology Laboratory, Public Health Agency of Canada, JC Wilt Infectious Diseases Research Centre, 745 Logan Avenue, Winnipeg, Manitoba, Postal Code R3E 3L5, Canada. Electronic address: Helene.schulz@phac-aspc.gc.ca.
² Viral Exanthemata and STD Section, National Microbiology Laboratory, Public Health Agency of Canada, JC Wilt Infectious Diseases Research Centre, 745 Logan Avenue, Winnipeg, Manitoba, Postal Code R3E 3L5, Canada. Electronic address: mackenzie.neale@phac-aspc.gc.ca.
³ Viral Exanthemata and STD Section, National Microbiology Laboratory, Public Health Agency of Canada, JC Wilt Infectious Diseases Research Centre, 745 Logan Avenue, Winnipeg, Manitoba, Postal Code R3E 3L5, Canada. Electronic address: vanessa.a.zubach@phac-aspc.gc.ca.
⁴ Viral Exanthemata and STD Section, National Microbiology Laboratory, Public Health Agency of Canada, JC Wilt Infectious Diseases Research Centre, 745 Logan Avenue, Winnipeg, Manitoba, Postal Code R3E 3L5, Canada; Department of Medical Microbiology and Infectious Diseases, Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address: alberto.severini@phac-aspc.gc.ca.
⁵ Viral Exanthemata and STD Section, National Microbiology Laboratory, Public Health Agency of Canada, JC Wilt Infectious Diseases Research Centre, 745 Logan Avenue, Winnipeg, Manitoba, Postal Code R3E 3L5, Canada. Electronic address: joanne.hiebert@phac-aspc.gc.ca.

PMID: 35217102
DOI: 10.1016/j.jviromet.2022.114500

Abstract

Rubella surveillance in elimination setting relies on rapid molecular detection of the virus. In this study a multiplex real-time RT-PCR assay for the detection of rubella virus was validated. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the rubella viral coding regions for envelope glycoprotein E1 and non-structural p150 protein, and an endogenous control (human RNaseP). Using dilution series of synthetic RNAs, the limits of detection were determined to be at least 50 copies of rubella RNA. The assay is reproducible with low intra-assay and inter-assay coefficients of variation for both the E1 and the p150 targets. After testing 62 confirmed rubella positive and 165 rubella negative archival clinical samples, the sensitivity and specificity of the multiplex assay were 98.4 and 100 %, respectively. No cross reactivity was identified with clinical specimens positive for eleven other viruses. This multiplex assay successfully detected nine viral genotypes including the predominant genotypes 1E, 1G, 1J, and 2B as well as the 1a vaccine genotype.

Keywords: Multiplex real-time RT-PCR; Rubella; Rubella surveillance.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Humans
RNA, Viral / genetics
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Rubella virus* / genetics
Rubella* / diagnosis
Rubella* / epidemiology
Sensitivity and Specificity

Substances

RNA, Viral