Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Abstract

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleicacid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

“…Currently, this is normally done by showing the presence of SARS-CoV-2 RNA in a frontal nose swab using a real-time RT-PCR (rRT-PCR) test 3 . Generally, these SARS-CoV-2 rRT-PCR test panels have several targets in one or more genes of the virus, complemented with several positive controls and negative controls for checking the correct execution of the whole procedure, including RNA extraction and RNA reverse-transcription efficacy (RT) 4 . Early 2020, the Division of Viral Disease of the Centers for Disease Control and Prevention (CDC, Atlanta, USA) has put a rRT-PCR diagnostic panel together for the detection of the 2019-Novel Coronavirus (https://www.fda.gov/media/134922/download).…”

Assessment of sample pooling for clinical SARS-CoV-2 testing

“…Not least among the latter has been a dramatic increase in sample testing load (1). Efforts to meet the demand have included increased use of automated instrumentation, multiplexing of molecular detection assays, streamlined testing protocols, as well as increasingly varied acceptable sample types, collection devices and transport media (2)(3)(4). Accommodating the testing workload and reagent shortage during the symptomatic pandemic wave was a significant undertaking (1).…”

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

“…Quantitative reverse-transcription (qRT)-PCR has become a critical tool for detecting SARS-CoV-2, by amplifying virus-derived RNA, due to its increased sensitivity and fast processing time (Esbin et al 2020) . However, this has been limited by the requirement for labor-intensive RNA extractions in order to enrich viral RNA.…”