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.

Efficacy of SARS-CoV-2 Vaccination in Dialysis Patients: Epidemiological Analysis and Evaluation of the Clinical Progress

“…In addition to the crucial role of vaccines, other factors have made an important contribution to the control of the initially distasteful effects of COVID-19 in dialysis patients. The experience acquired over time since the first pandemic phases paved the way for the collaboration between interdisciplinary teams [ 44 ], the adoption of targeted measures (dedicated rooms, dedicated pathways) [ 45 ] and the development of earlier and more sensitive diagnostic tests [ 46 , 47 ], together with the pharmacological research on novel therapeutic agents [ 48 , 49 ].…”

Temporary Regulatory Deviations and the Coronavirus Disease 2019 (COVID-19) PCR Labeling Update Study Indicate What Laboratory-Developed Test Regulation by the US Food and Drug Administration (FDA) Could Look Like

“…The labeling update experiments were performed using Massachusetts General Hospital's EUA real-time PCR assay for detection of SARS-CoV-2 RNA. 41 Briefly, the assay targets SARS-CoV-2 N1 , N2 , and human RNaseP in separate wells. The cost analysis included nucleic acid extraction using the Total Nucleic Acid Isolation kit on the MagNA Pure 24 instrument (Roche, Basel, Switzerland), followed by PCR using the 2 (N1-/N2-) SARS-CoV-2 specific primer and probe mix (IDT, Coralville, IA) and TaqPath 4× Master Mix on a QuantStudio 7 real-time PCR instrument (ThermoFisher Scientific, Waltham, MA).…”

Shine: A novel strategy to extract specific, sensitive and well-conserved biomarkers from massive microbial genomic datasets

“…The testing and rapid detection of pathogenic organisms is a crucial undertaking related to health, safety and wellbeing, especially for the early detection of pathogens, which is important for diagnosing and preventing diseases [1][2][3]. 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 [4]. Obviously, the process of developing an emergency-use molecular-based laboratory-developed test (LDT) would be useful to other laboratories in future outbreaks and would help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions [4].…”

“…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 [4]. Obviously, the process of developing an emergency-use molecular-based laboratory-developed test (LDT) would be useful to other laboratories in future outbreaks and would help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions [4]. Nevertheless, the deoxyribonucleic acid (DNA) concentrations of pathogenic microorganisms in biological samples are mostly very low and close to the detection limit, so pathogen detection has become one of the most challenging aspects in clinical applications [5].…”