Development of a qualitative real-time RT-PCR assay for the detection of SARS-CoV-2: A guide and case study in setting up an emergency-use, laboratory-developed molecular assay

Abstract

Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

“…To detect SARS-CoV-2 using RT-LAMP, we took advantage of the primer set previously validated in vitro by Zhang et al . 24 and tested, on clinical specimens from large cohorts of COVID-19 patients, by several other authors 16 – 19 . The primer set (N-A) targeted the N gene, which encodes the nucleocapsid protein and has the most abundant expression of subgenomic mRNA during infection 32 – 35 .…”

“…Several authors have recently shown that the use of different primer sets boosts RT-LAMP sensitivity, possibly due to better primer efficiency and/or higher target abundance. Also different saliva treatment protocols, combining certain chemicals and proteinase K, have been shown to improve SARS-CoV-2 detection in saliva samples 16 , 21 , 22 , 28 , 29 , 31 . Thus, we reason that there is still room to improve the sensitivity of our test.…”

Evaluation of the diagnostic accuracy of a new point-of-care rapid test for SARS-CoV-2 virus detection

“…Laboratory real-time (RT)-PCR represents the standard of care for the detection of the SARS-CoV-2 infection [6,7], but this technique is time-consuming (up to 24 h for the result), not always available, and the technical requirements usually can only be met by centralized diagnostic laboratories.…”

Evaluation of the diagnostic accuracy of a new point-of-care rapid test for SARS-CoV-2 virus detection

“…Laboratory real-time (RT)-PCR represents the standard of care for the detection of the SARS-CoV-2 infection [6,7], but this technique is time-consuming (up to 24 hours for the result), not always available, and the technical requirements usually can only be met by centralized diagnostic laboratories.…”

“…Different diagnostic strategies are now available to identify or exclude a current infection, identify people in need of care escalation, or to test for past infection and immune response [4,5]. Laboratory real-time (RT)-PCR represents the standard of care for the detection of the SARS-CoV-2 infection [6,7], but this technique is time-consuming (up to 24 hours for the result), not always available, and the technical requirements usually can only be met by centralized diagnostic laboratories.…”