Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology
Garrett A PerchettiAlexander L Greninger

Abstract

The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the tripl...Continue Reading

Citations

Oct 30, 2020·International Journal of Infectious Diseases : IJID : Official Publication of the International Society for Infectious Diseases·Byron Freire-Paspuel, Miguel Angel Garcia-Bereguiain
Dec 15, 2020·Journal of Virological Methods·Nefeli LagopatiVassilis G Gorgoulis
Jan 26, 2021·Trends in Analytical Chemistry : TRAC·Jiri KudrOndrej Zitka
Feb 26, 2021·BMJ Open Respiratory Research·Souichi YamadaUNKNOWN Virus Diagnosis Group (NIID Toyama)
Mar 19, 2021·Journal of Medical Microbiology·Jamie L ThompsonClaire H Seedhouse
Jul 1, 2021·Journal of Clinical and Translational Science·Jeffery H MoranLaura James
Jul 22, 2021·Current Issues in Molecular Biology·Ahmad QasemSaleh A Naser
Jul 24, 2021·The Science of the Total Environment·Anna NavarroTeresa Lettieri
Aug 22, 2021·International Journal of Biological Macromolecules·Mehrnaz EbrahimiAli Akbar Moosavi-Movahedi
Oct 16, 2021·Scientific Reports·Kristína BoršováBoris Klempa

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Methods Mentioned

BETA
reverse transcription PCR
PCR
PCRs

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