A rapid RT-LAMP SARS-CoV-2 screening assay for collapsing asymptomatic COVID-19 transmission

Abstract

Purpose: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population.

Methods: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples.

Results: Validation samples reporting a Ct < 20 were detected at 100% DSe and DSp, reducing to 95% DSe (100% DSp) for all samples reporting a Ct < 30 (both genomic dual sub-genomic assays). Advisory screening identified nine positive cases in 1680 symptom free individuals (equivalent to 540 cases per 100,000) with results reported back to participants and feed into national statistics within 48 hours.

Conclusion: This work demonstrates the utility of a rapid RT-LAMP assay for collapsing transmission of SARS-CoV-2 in an asymptomatic screening population.

Fig 1. University of Leicester SARS-CoV-2 advisory screening programme.

Available to all students and staff without symptoms for a period of twelve weeks (October 2020 –December 2020) to allow rapid isolation and reduce outbreaks.

Fig 2. RT-LAMP primer investigation.

SARS-CoV-2 RNA primer sets targeting the nucleopcapsid (N), envelope gene (E) (NEB design) and Orf1a (Rabe and Cepko Harvard Medical School) were tested independently and in combination (dual N+E reaction and multiplex Orf1a+N+E reaction) against 1x10⁴ copies of Twist synthetic SARS-CoV-2 control RNA (T1 and T2). Negative control RNA from Betacoronavirus 1 strain OC43 and Influenza A (H1N1) at a single concentration (1x10⁵ copies per well) plus a water no template control (NTC). A total RNA control primer set (NEB) targeting human beta actin (ACTB) was also included and tested against 5 ng total human RNA (hRNA). Both RT-LAMP fluorescent end-point and colorimetric 25 μl reactions were performed at 65°C for 40 minutes on a StepOnePlus thermoclycler. (A) Representative fluorescent amplification curves where time to positive (TTP) is the time at which amplification exceeds the manually set, reaction consistent threshold (red dotted line). (B) Representative colorimetric reactions whereby yellow indicates positive amplification and pink no amplification.

Fig 3. Limit of detection of fluorescent end-point RT-LAMP reactions targeting genomic and sub-genomic regions of SARS-CoV-2.

Twist Bioscience synthetic positive control RNA (control 2 GenBank ID MN908947.3, GISAID Wuhan-Hu-1) was serially diluted to 10,000, 1,000, 500, 100, 50 and 10 copies of viral sequence per 25 μl reaction. Water no template control (NTC) were included in each reaction. Reactions were performed at 65°C for 40 minutes on the Qiagen Rotor-Gene Q Thermoclycler platform. Representative amplification and linear regression analysis for each primer set are shown. (A) RT-LAMP targeting the Orf1a. (B) RT-LAMP targeting N+E duplex. (C) RT-LAMP triple Orf1a+N+E target. Time to positive (TTP) is the time at which amplification exceeds the manually set, reaction consistent threshold (red dotted line) when amplification enters the rapid linear, exponential phase. Data represents the average of two experiments each performed in quadruplicate.