Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR
| Autor: | Garrett A. Perchetti, Alexander L. Greninger, Arun K. Nalla, Keith R. Jerome, Meei-Li Huang |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 030106 microbiology Pneumonia Viral RT-PCR Quantitative Reverse Transcriptase PCR Biology Real-Time Polymerase Chain Reaction Multiplexing Sensitivity and Specificity Article 03 medical and health sciences Betacoronavirus 0302 clinical medicine COVID-19 Testing Virology LDT Humans Multiplex 030212 general & internal medicine Pandemics DNA Primers Clinical Laboratory Techniques SARS-CoV-2 virus diseases Respiratory infection COVID-19 Infectious Diseases Real-time polymerase chain reaction Triplex Respiratory virus RNA Viral Primer (molecular biology) Coronavirus Infections Oligonucleotide Probes |
| Zdroj: | Journal of Clinical Virology |
| ISSN: | 1873-5967 |
| Popis: | Highlights • Multiplexing three targets in a single-reaction maintains LDT assay performance. • Triplexing correctly identified SARS-CoV-2 in 98.4% positive or inconclusive samples. • Primers and probes used in multiplexed assays are virus specific. • Of all 356 samples tested, triplexing demonstrated 99.2% (n = 353/356) assay agreement. 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. |
| Databáze: | OpenAIRE |
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