Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.

Laverack M ，Tallmadge RL ，Venugopalan R ，Cronk B ，Zhang X ，Rauh R ，Saunders A ，Nelson WM ，Plocharczyk E ，Diel DG ... -  《-》

A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

Bland J ，Kavanaugh A ，Hong LK ，Perez O ，Kadkol SS ... -  《-》

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR.

de Kock R ，Baselmans M ，Scharnhorst V ，Deiman B ... -  《-》

Comparing two sample pooling strategies for SARS-CoV-2 RNA detection for efficient screening of COVID-19.

The emerging pandemic of coronavirus disease 2019 (COVID-19) has affected over 200 countries and resulted in a shortage of diagnostic resources globally. Rapid diagnosis of COVID-19 is vital to control the spreading of the disease, which, however, is challenged by limited detection capacity and low detection efficiency in many parts of the world. The pooling test may offer an economical and effective approach to increase the virus testing capacity of medical laboratories without requiring more laboratory resources such as laboratory workers, testing reagents, and equipment. In this study, the sample pools of 6 and 10 were detected by a real-time reverse transcription-polymerase chain reaction assay targeting ORF1ab and N genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Each pool consisted of five or nine negative SARS-CoV-2 samples and one positive counterpart with varying viral loads. Two different strategies of sample pooling were investigated and the results were compared comprehensively. One approach was to pool the viral transport medium of the samples in the laboratory, and the other was to pool swab samples during the collection process. For swab pooling strategy, qualitative results of SARS-CoV-2 RNA, specific tests of ORF1ab and N genes, remained stable over the different pool sizes. Together, this study demonstrates that the swab pooling strategy may serve as an effective and economical approach for screening SARS-CoV-2 infections in large populations, especially in countries and regions where medical resources are limited during the pandemic and may thus be potential for clinical laboratory applications.

Chen F ，Geng Z ，Wang J ，Liuchang W ，Huang D ，Xu Y ，Wang Z ，Wang L ... -  《-》

Development and Evaluation of AccuPower COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples.

Suh IB ，Lim J ，Kim HS ，Rhim G ，Kim H ，Kim H ，Lee SM ，Park HS ，Song HJ ，Hong M ，Shin GS ，Kim MJ ... -  《PLoS One》