SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay

Abstract

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

Reverse-Transcription Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals

“…Direct detection negates the requirement for RNA extraction 32 , 33 , for which there has previously been competition for reagents and often requires expensive extraction equipment including liquid handling automation. This extraction-free method decreases turnaround time from sample collection to result.…”

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management

“…Choice of the lysis buffers, for example, can yield vastly variable results, depending on its components 42 and reported success of RNA-extraction versus no-RNA-extraction results are contradictory. Untreated nasopharyngeal samples are likely to contain RNases 61 that would need to be inactivated prior to lysis and while Myhrvold and colleagues developed a nuclease inactivation and viral lysis method (HUDSON) to omit an extra RNA extraction step 72 and Ladha and co-workers condensed their isolation protocol down to a 5-min procedure 73 other studies claim the sample processing step to be unnecessary 12,74,75 . Furthermore, as there are still 5 steps that require user interventions, our protocol is prone to RNase contaminations and errors due to misuse.…”

“…Patients experiencing these symptoms, but also asymptomatic individuals (incidence ranging from 17.9 to 39.8% 5,6 ), whose timeframe of viral shedding can even exceed the symptomatic carrier’s 7 , substantially contribute to transmission 4,8–10 via respiratory droplets, fomites, direct or indirect contact 11 . Identification of these individuals via RT-qPCR, however, is difficult since the method is not necessarily suited for high-throughput analysis and difficult to implement in resource-limited environments 12 . Other drawbacks are comparability between labs 13,14 , an overestimation of infectious virions 15 , and a rather lengthy sample processing time of approximately 4-6 hours.…”

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control

“…Since the onset of the pandemic, a large number of publications emerged describing the use and validation of RT-LAMP assays for SARS-CoV-2 diagnostics, i.e. (20,27,(38)(39)(40)(41)(42)(43)(44). These publications describe many different primer sets and use patient samples without purification ("direct assays") (some are summarized here: (42)) or with prior purification of RNA, many with optimized procedures where compelling data could be obtained that suggest suitable sensitivity of RT-LAMP for detection of infections in asymptomatic and symptomatic subjects.…”