Development and validation of a multiplex reverse transcriptase-PCR assay for simultaneous testing of influenza A, influenza B and SARS-CoV-2

Kanti Pabbaraju; Anita A Wong; Raymond Ma; Nathan Zelyas; Graham A Tipples

doi:10.1016/j.jviromet.2021.114151

Development and validation of a multiplex reverse transcriptase-PCR assay for simultaneous testing of influenza A, influenza B and SARS-CoV-2

J Virol Methods. 2021 Jul:293:114151. doi: 10.1016/j.jviromet.2021.114151. Epub 2021 Apr 8.

Authors

Kanti Pabbaraju¹, Anita A Wong², Raymond Ma², Nathan Zelyas³, Graham A Tipples⁴

Affiliations

¹ Alberta Precision Laboratories, Public Health Laboratory, Calgary, Alberta, Canada. Electronic address: kanti.pabbaraju@albertaprecisionlabs.ca.
² Alberta Precision Laboratories, Public Health Laboratory, Calgary, Alberta, Canada.
³ Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Alberta, Canada; Alberta Precision Laboratories, Public Health Laboratory, Edmonton, Alberta, Canada; Department Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
⁴ Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Alberta, Canada; Alberta Precision Laboratories, Public Health Laboratory, Edmonton, Alberta, Canada; Department Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.

Abstract

In the current pandemic of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the co-circulation of SARS-CoV-2 and other respiratory viruses during the upcoming fall and winter seasons may present an unprecedented burden of respiratory disease in the population. Important respiratory viruses that will need to be closely monitored during this time include SARS-CoV-2, influenza A and influenza B. The epidemiology of these viruses is very similar in terms of susceptible populations, mode of transmission, and the clinical syndromes, thus the etiological agent will be difficult to differentiate without target specific assays. The availability of a sensitive and specific multiplex assay that can simultaneously detect all these targets will be valuable. Here we report the validation of a real-time reverse transciptase-PCR assay for the simultaneous detection of SARS-CoV-2, influenza A and influenza B. This multiplex assay is comparable to its singleplex counterparts with a limit-of-detection being less than 5 copies/reaction, 100 % specificity, over seven logs of dynamic range, less than 1 % coefficientof variation showing high precision, and equivalent accuracy using patient samples. It also offers the added benefits of savings in reagents and technologist time while improving testing efficiency and turn-around-times in order to respond effectively to the ongoing pandemic.

Keywords: COVID-19; Co-infection; Influenza; SARS-CoV-2; multiplex.

Publication types

Validation Study

MeSH terms

COVID-19 / diagnosis*
COVID-19 Nucleic Acid Testing / methods*
Coinfection / diagnosis
Humans
Influenza A virus / genetics*
Influenza B virus / genetics*
Influenza, Human / diagnosis*
Multiplex Polymerase Chain Reaction / methods*
Reproducibility of Results
SARS-CoV-2 / genetics*
Sensitivity and Specificity