Development and validation of RT-PCR assays for testing for SARS-CoV-2.

Background: The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays. Methods: We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control. Results: These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention. Conclusions: The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.

Historique: La récente émergence et la propagation mondiale rapide du coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) a démontré l'urgence de créer des dosages en laboratoire pour poser un diagnostic clinique et adopter des interventions sanitaires en l'absence de dosages commerciaux. Méthodologie: Les chercheurs exposent la progression des dosages d'amplification en chaîne par polymérase couplée à la transcriptase inverse (RT-PCR) mis au point et validés par les Alberta Precision Laboratories du Laboratoire de santé publique de l'Alberta, au Canada, pour répondre à cette pandémie. Les tests ont d'abord été effectués au moyen de dosages sur gel spécifiques au SARS-CoV-2 ou décelant tous les coronavirus, mais ont vite été remplacés par des dosages RT-PCR en temps réel ciblant l'enveloppe et les gènes d'ARN polymérase sous la dépendance d'ARN pour répondre au fort volume anticipé d'échantillons. Le criblage a également été renforcé par le multiplexage conjoint des différentes cibles et la codétection d'un contrôle d'extraction interne. Résultats: Ces dosages ont une sensibilité et une spécificité comparables à ceux recommandés par l'Organisation mondiale de la Santé et les Centers for Disease Control and Prevention des États-Unis. Conclusions: Il était essentiel de disposer de dosages RT-PCR au début de la pandémie pour que les autorités sanitaires locales puissent profiter de temps précieux pour contenir la transmission et préparer les stratégies de réponse appropriées.

Development and validation of a multiplex reverse transcriptase-PCR assay for simultaneous testing of influenza A, influenza B and SARS-CoV-2.

In the current pandemic of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the co-circulation of SARS-CoV-2 and other respiratory viruses during the upcoming fall and winter seasons may present an unprecedented burden of respiratory disease in the population. Important respiratory viruses that will need to be closely monitored during this time include SARS-CoV-2, influenza A and influenza B. The epidemiology of these viruses is very similar in terms of susceptible populations, mode of transmission, and the clinical syndromes, thus the etiological agent will be difficult to differentiate without target specific assays. The availability of a sensitive and specific multiplex assay that can simultaneously detect all these targets will be valuable. Here we report the validation of a real-time reverse transciptase-PCR assay for the simultaneous detection of SARS-CoV-2, influenza A and influenza B. This multiplex assay is comparable to its singleplex counterparts with a limit-of-detection being less than 5 copies/reaction, 100 % specificity, over seven logs of dynamic range, less than 1 % coefficientof variation showing high precision, and equivalent accuracy using patient samples. It also offers the added benefits of savings in reagents and technologist time while improving testing efficiency and turn-around-times in order to respond effectively to the ongoing pandemic.

A Public Health Laboratory Response to the Pandemic.

An outbreak of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) began in Wuhan, Hubei, China, in December 2019 and spread rapidly worldwide. The response by the Alberta Precision Laboratories, Public Health Laboratory (ProvLab), AB, Canada, included the development and implementation of nucleic acid detection-based assays and dynamic changes in testing protocols for the identification of cases as the epidemic curve increased exponentially. This rapid response was essential to slow down and contain transmission and provide valuable time to the local health authorities to prepare appropriate response strategies. As of May 24, 2020, 236,077 specimens were tested, with 6,475 (2.74%) positives detected in the province of Alberta, Canada. Several commercial assays are now available; however, the response from commercial vendors to develop and market validated tests is a time-consuming process. In addition, the massive global demand made it difficult to secure a reliable commercial supply of testing kits and reagents. A public health laboratory serves a unique and important role in the delivery of health care. One of its functions is to anticipate and prepare for novel emerging pathogens with a plan for pandemic preparedness. Here, we outline the response that involved the development and deployment of testing methodologies that evolved as SARS-CoV-2 spread worldwide, the challenges encountered, and mitigation strategies. We also provide insight into the organizational structure of how a public health response is coordinated in Alberta, Canada, and its benefits.