DEVELOPMENT AND ANALYTICAL VALIDATION OF COLORIMETRIC DUPLEX RT-LAMP ASSAYS FOR RAPID DETECTION OF SARS-CoV-2 RNA IN CLINICAL SAMPLES

A. MUÑOZ-CALDERÓN ; M. AVARO; S. BESUSCHIO; S. LONGHI; G. CABRERIZO; M. APODACA; A. PONTORIERO; F. REMES LENICOV; D. WEHRENDT; S. VILCHES; E. BENEDETTI; G. RODRIGUEZ; A. SCHIJMAN

Virtual

Congreso; Reunión de Sociedades de Biociencias 2020; 2020

SAIC - SAFICI -SAI

AIMS To evaluate duplex RT-LAMP for detection of SARS-CoV-2 RNA.METHODS i) 2 Sets of primers (6 primers/set) for each ORF1ab, Nucleocapsid(N) and Spike(S) viral genes plus 2 sets for human RNASeP gene as control were designed and tested in silico for inclusivity against sequences from strains circulating in Argentina and abroad and exclusivity against those from related viruses. We used NEB RT-LAMP kit in simplex or duplex formats, plus a RT-LAMP for sample integrity control, using RNAs from RT-PCR tested nasopharyngeal swabs and synthetic viral RNAs. ii) LOD95 was calculated for duplex RT-LAMP tests following FDA and WHO COVID-19 guidelines, using a quantified SARS-CoV-2 positive RNA control (GISAID EPI_ISL_420600) isolated from a local viral strain cultured in Vero cells, and quantified against a WHO SARS-CoV-2 standard. iii) RT-LAMP visualization was done by naked eye and by a mobile app. Strips were photographed using a simple capture device with visual references to standardize strip-tubes positions, focus and light intensity, and colour analysis was implemented by computer vision techniques. iv) a blind panel of RNAs from 20 qPCR positive and 21 qPCR negative samples was tested. Positivity was visualized by colour change between 30-40 min. of incubation at 65°C. Kappa agreement between RT-LAMP and RT-PCR was estimated.RESULTS LOD95 of selected duplex RT-LAMPS (ORF+N, N+S and S+ORF) ranged between 12.5-15 RNA copies/reaction. The mobile phone app reported correctly photographs of tested strips in accordance with naked-eye visualization and reporting. All 41 swabs were RNASeP-RT-LAMP positive. Maximum Kappa indexes between RT-LAMP and RT-PCR were excellent: 0.902 for ORF+S at 30 min (95% Se&Sp) and 0.804 for S+N at 30 min and ORF+N at 40 min (80%Se,100%Sp).CONCLUSIONS Optimization of rapid RNA extraction methods from clinical samples and prospective blind-based evaluation is a next step towards validating these tools for point-of-care COVID-19 diagnosis.