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

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 triplex reaction. 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.

Perchetti GA ，Nalla AK ，Huang ML ，Jerome KR ，Greninger AL ... -  《-》

Validation and verification of the Abbott RealTime SARS-CoV-2 assay analytical and clinical performance.

High-throughput assays for the SARS-CoV-2 virus are critical to increasing test capacity and slowing the spread of COVID-19. Abbott Molecular developed and received emergency use authorization (EUA) to deploy the new RealTime SARS-CoV-2 assay, run on the automated m2000sp/rt system. To evaluate analytical and clinical performance of the RealTime SARS-CoV-2 assay compared to the SARS-CoV-2 CDC-based laboratory developed test (LDT) in clinical use by the University of Washington Clinical Virology Laboratory (UW Virology). RealTime SARS-CoV-2 assay limit of detection (LOD) was evaluated by testing two dilution panels of 60 replicates each. Cross-reactivity was evaluated by testing 24 clinical samples positive for various non‒SARS-CoV-2 respiratory viruses. Clinical performance was evaluated using 30 positive and 30 negative SARS-CoV-2 clinical samples previously tested using the UW Virology SARS-CoV-2 LDT. Exceeding the 100 copies/mL LOD reported in the RealTime SARS-CoV-2 assay EUA product insert, 19 of 20 replicates were detected at 50 copies/mL and 16 of 20 replicates were detected at 25 copies/mL. All clinical samples positive for 24 non‒SARS-CoV-2 respiratory viruses were SARS-CoV-2 negative on the RealTime SARS-CoV-2 assay. The assay had high sensitivity (93%) and specificity (100%) for detecting SARS-CoV-2 in clinical samples. Two positive samples that tested negative with the RealTime SARS-CoV-2 assay had cycle numbers of 35.94 or greater and required dilution prior to testing. One of these samples was also inconclusive on the SARS-CoV-2 LDT. The RealTime SARS-CoV-2 assay is acceptable for clinical use. With the high-throughput, fully automated m2000 system, this assay will accelerate the pace of SARS-CoV-2 testing.

Degli-Angeli E ，Dragavon J ，Huang ML ，Lucic D ，Cloherty G ，Jerome KR ，Greninger AL ，Coombs RW ... -  《-》

Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline.

During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.

Kuchinski KS ，Jassem AN ，Prystajecky NA 《-》

Comparison of Commercially Available and Laboratory-Developed Assays for In Vitro Detection of SARS-CoV-2 in Clinical Laboratories.

Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 (Cepheid, DiaSorin, Hologic Panther, and Roche Cobas) on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 failed to detect positive specimens only near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 reverse transcription-PCR (RT-PCR) and highlight the importance of having multiple viral detection testing platforms available in a public health emergency.

Lieberman JA ，Pepper G ，Naccache SN ，Huang ML ，Jerome KR ，Greninger AL ... -  《-》

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit.

Nearly 400,000 people worldwide are known to have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beginning in December 2019. The virus has now spread to over 168 countries including the United States, where the first cluster of cases was observed in the Seattle metropolitan area in Washington. Given the rapid increase in the number of cases in many localities, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we evaluated assays using seven different primer-probe sets and one assay kit. We found that the most sensitive assays were those that used the E-gene primer-probe set described by Corman et al. (V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25:2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set developed by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer-probe set or kit used. These results will provide valuable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.

Nalla AK ，Casto AM ，Huang MW ，Perchetti GA ，Sampoleo R ，Shrestha L ，Wei Y ，Zhu H ，Jerome KR ，Greninger AL ... -  《-》