








key: cord-340336-u59l0taa
authors: Perchetti, Garrett A.; Nalla, Arun K.; Huang, Meei-Li; Jerome, Keith R.; Greninger, Alexander L.
title: Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR
date: 2020-06-08
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104499
sha: 
doc_id: 340336
cord_uid: u59l0taa

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.

 -Of all 356 samples tested, triplexing demonstrated 99.2% (n=353/356) assay agreement 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.

The novel virus responsible for causing coronavirus disease 2019 (COVID-19), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has infected more than six million individuals in 188 countries as of writing [1] . Emerging from Wuhan, China in late 2019, the ongoing pandemic has been intensified by lack of adequate diagnostic testing in the US and internationally [2] . SARS-CoV-2 is highly communicable with significant morbidity and mortality [3, 4, 5] . Early detection of SARS-CoV-2 can identify patients who are more likely to experience significant disease and so curb pathogen transmission and scope of global contagion.

Many labs use the Centers for Disease and Control and Prevention (CDC) primer and probe sets targeting N1 and N2 for SARS-CoV-2 and RPP30 as a human control [6] . As the CDC kits utilize the same fluorescent reporter for each of the primer/probe sets, reactions are required to be run separately, leading to fewer than 30 samples per 96-well plate. To increase throughput of SARS-CoV-2 testing in clinical laboratories, we designed a multiplexed real-time quantitative reverse transcription PCR (qRT-PCR) assay utilizing primers and probe sets from the CDC combined with an internal extraction control.

Multiplexed qRT-PCR is a powerful tool in laboratory medicine, able to detect infectious disease pathogens effectively and efficiently. Multiple target assays are critical for accurate SARS-CoV-2 detection, as it is possible to miss low viral load infections if only a single gene amplicon is used. After running a duplex reaction with N1 and N2 in separate wells with internal control, we developed a three-target single-reaction triplex assay with the same viral nucleocapsid gene targets. Multiplexing offers increased throughput of SARS-CoV-2 detection by reducing the quantity of qRT-PCR reactions run in parallel [7] . Here, we describe a singlereaction, triplex assay for SARS-CoV-2 that demonstrates comparable sensitivity to individual parallel assays.

The SARS-CoV-2 positive control consisted of a wild-type clinical nasopharyngeal (NP) swab tested at UW Virology in late February, 2020. HeLa cells for extraction and no template controls of water for amplification were included as negative standards. NP swabs in viral transport media were submitted to UW Virology for COVID-19 clinical testing by LDT beginning in March 2020. Specimens were subsequently compared to triplex assay performance by CTs and percent of positive samples detected.

Nucleic acid (NA) extraction was performed on Roche's MagNA Pure 96 instrument enabling high-throughput total NA extraction using the pathogen universal kit [8] . In brief, 200µL of sample was extracted and eluted into 50µL elution buffer and 5µL of eluted template was utilized for each subsequent 25µL LDT assay, whereas 11µL of eluted RNA was used for triplexing.

Distinct amplicons within the N gene, the region encoding a nucleocapsid protein of SARS-CoV-2, were targeted for detection: N1 and N2. Each target was combined with EXO (a 130-base RNA transcript derived from jellyfish DNA) to serve as an internal extraction control [9, 10] . If all targets amplified, the result was determined positive. If only one of the N gene targets amplified with EXO, then the result is inconclusive and subsequently re-tested. Not detected (NDET) test results required the amplification of EXO, without N1 or N2 amplification. reaction, the threshold was set at 0.025 for N1 and N2, while EXO's threshold was set at 0.02 [13] .

We compared the performance of a novel triplex three-target assay run in parallel to the Washington state emergency use authorization LDT performed at UW Virology. Positive Multiplexing offers a two-reaction or even single-reaction assay that significantly reduces reagent consumption, labor, inconsistencies in reporting, and frees up valuable lab equipment when it is critically needed. We demonstrated that the CDC individual assays can be triplexed into a single-reaction without substantially compromising sensitivity, detecting 98.4% of samples determined positive or inconclusive by SARS-CoV-2 LDT.

This work was supported by the department of Laboratory Medicine at the University of Washington Medical Center.

The authors declare no conflict of interest.

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We would like to acknowledge all essential workers in healthcare and the front lines during the COVID-19 pandemic. We would also like to thank Reigran Sampoleo for assistance with EXO sequence data.

