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 ... -  《-》

Comparison of SARS-CoV-2 PCR-Based Detection Using Saliva or Nasopharyngeal Swab Specimens in Asymptomatic Populations.

Fan G ，Qin X ，Streblow DN ，Hoyos CM ，Hansel DE ... -  《Microbiology Spectrum》

Saliva is Comparable to Nasopharyngeal Swabs for Molecular Detection of SARS-CoV-2.

Callahan C ，Ditelberg S ，Dutta S ，Littlehale N ，Cheng A ，Kupczewski K ，McVay D ，Riedel S ，Kirby JE ，Arnaout R ... -  《Microbiology Spectrum》

Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2.

Margulis M ，Rohana H ，Erster O ，Mandelboim M ，Biber A ，Schwartz E ，Peretz A ，Danielli A ... -  《-》

Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples.

Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

Cassinari K ，Alessandri-Gradt E ，Chambon P ，Charbonnier F ，Gracias S ，Beaussire L ，Alexandre K ，Sarafan-Vasseur N ，Houdayer C ，Etienne M ，Caron F ，Plantier JC ，Frebourg T ... -  《-》