ACTIVE SURVEILLANCE OF SARS CoV-2 DELTA VARIANT BY RT-qPCR ON POOLED SAMPLES

MANZUR MARÍA JIMENA; CAMPOS LUDMILA; LEPORATI MARIANELA; ARIAS JOSÉ LUIS; JURI AYUB MAXIMILIANO

san luis

Congreso; XXXIX Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2021

Emergence of variants of concern (VOCs) during SARS-Cov 2 evolution has generated stressed worldwide health systems and an increase in the number of hospitalizations and deaths. The gold standard approach for variants surveillance is whole or partial genome sequencing. However, this tool is expensive and time consuming, limiting the number of samples that can be analyzed. Thus, faster and cost-effective strategies for VOCs identification are essential for efficient decision making. Therefore, we have developed protocols for specific identification of all current VOCs (a, b, g and d) as well as the Variant of Interest (VOI) l by RT-qPCR, which is broadly accessible and can be implemented in any diagnostic laboratory. In the present work, we describe a protocol that has been successfully implemented for early detection of VOCd community circulation, as well as rapid testing of travelers. Based on genomic data, we designed a pooled RT- qPCR detecting specifically nucleotides 11,288-11,296 of SARS Cov-2 genome (encoding amino acids 3675-3677 of ORF1ab protein). This 9 bp segment is absent in variants a, b, g and l, which together currently represent more than 99% of circulation in our country, but is present in VOCd. Therefore, if ORF1ab target amplification is detected, samples of the positive pool are individually analyzed by a second, confirmatory PCR in which a probe covering the VOCd-specific deletion (nucleotides 22,029-22,034) is used. Asymmetric PCR followed by probe melting assay yields melting peaks of 40°C and 60°C for VOCd and other lineages, respectively. This approach has been applied to more than 1,000 samples in several provinces (including San Luis and Mendoza). Form these, 13 samples were identified as VOCd using this protocol. From these, four samples have been further confirmed by sequencing at PAIS project, yielding 100 % of concordance. Thus, our multiplex RT-qPCR variant screening assay is a useful tool to prioritize samples for sequencing and as powerful surveillance tool for decision-making instrument by governments.