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 microbiological assay

Abstract

Developing and deploying new diagnostic tests are difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important. During a pandemic, laboratories play a key role in helping healthcare 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, 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 establishing fast and accurate diagnostic testing in crisis conditions.

Keywords: diagnosis; diagnostic techniques and procedures; laboratory manual; microbiology; virology.

Figure 1

A timeline of events placing the development of the LDT assay in the context of the local epidemic of COVID-19. Histogram showing the showing the local epidemic curve as defined by daily cases reported to the state Public Health Laboratory. Key LDT development milestones are shown below. CDC, Centers for Disease Control and Prevention; EUA, Emergency Use Authorization; FDA, Food and Drug Administration; LDT, laboratory developed test; LoD, limit of detection.

Figure 2

Clinical performance assessment in COVID-positive specimens, contrived positive specimens and negative controls. Ct (cycle threshold) values for N1, N2, and RNAse P primer-probe sets are shown. A no template control (NTC) is shown in blue. Contrived positive nasopharyngeal (NP) specimens are represented in yellow, at four different concentrations (from 10 to 10,000 SARS-CoV-2 copies/µL of sample). Clinical samples, at the right of the figure, comprise known COVID-negative samples (green) and COVID-positive samples (red). N1 and N2 represent reactions with SARS-CoV-2 specific primer pairs, with primer sequences consistent with those published by the CDC. RNase P primers amplify human RNA and thus these reactions serve as positive controls to ensure that these specimens do not contain significant PCR inhibitors and have adequate sample quality. CDC, Centers for Disease Control and Prevention.