Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.
J Clin Virol
; 129: 104499, 2020 08.
Article
in English
| MEDLINE | ID: covidwho-574871
ABSTRACT
BACKGROUND:
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.METHODS:
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.RESULTS:
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 triplex reaction.CONCLUSIONS:
Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Pneumonia, Viral
/
RNA, Viral
/
Oligonucleotide Probes
/
DNA Primers
/
Coronavirus Infections
/
Clinical Laboratory Techniques
/
Real-Time Polymerase Chain Reaction
/
Betacoronavirus
Type of study:
Diagnostic study
/
Experimental Studies
/
Prognostic study
/
Randomized controlled trials
Limits:
Humans
Language:
English
Journal:
J Clin Virol
Journal subject:
Virology
Year:
2020
Document Type:
Article
Affiliation country:
J.jcv.2020.104499
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