An Upgrade on the Surveillance System of SARS-CoV-2: Deployment of New Methods for Genetic Inspection

Abstract

SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.

Prevalence of symptoms, comorbidities, and reinfections in individuals infected with Wild-Type SARS-CoV-2, Delta, or Omicron variants: a comparative study in western Mexico

“…Following diagnosis, positive SARS-CoV-2 samples with a Ct-value of <27 of the N2 region gene were processed by the RT-qPCR Master Mut Kit (Genes2Life, Mexico), which has been validated as a rapid SARS-CoV-2 VOC screening test ( 20 ). This assay was performed according to the manufacturer’s instructions.…”

“…Nevertheless, this approach can result in expensive, slow, and complex processes because of the highly qualified personnel required for its fulfillment. In addition, the economic gap has made this difficult for developing countries such as Mexico to rely on these technologies entirely; therefore, other validated quantification methods for variant detection, such as screening SNP assays via RT-qPCR technology, have been extensively employed ( 20 , 21 ).…”

Bridging the Sequencing Gap: N501Y SNP RT-qPCR Assay Detects First SARS-CoV-2 Beta Variant in the Philippines

“…This includes microbiological risk definition using S-dropout as an indicator for the Alpha lineage, and the establishment of N501Y-specific RT-PCR assays (15). Another research initiative opted to use an RT-qPCR screening test, facilitating the analysis of a larger sample size within a shorter timeframe and at a reduced cost (16). They identified variants in samples positive for SARS-CoV-2 by an RT-qPCR mutation screening kit and subsequently confirmed through Next Generation Sequencing (NGS).…”

“…An RT-qPCR assay that targets variant-specific SNP mutations like N501Y can be used as a screening tool for early variant classification (11). Laboratory-developed SNP RT-qPCR assays were implemented in the surveillance measures in several countries, which allowed them to screen and identify probable variants rapidly (13)(14)(15)(16)(17). In this paper, we optimized and used a SNP RT-qPCR assay to screen for the variant-associated N501Y amino acid substitution in banked SARS-CoV-2 samples.…”

Vitamin A Positively Correlates with Secretory Immunoglobulin A: A Cross-Sectional Study in Omicron COVID-19 Outpatients

“…Confirmation of SARS-CoV-2 was established through RT-PCR using the DeCoV19 Kit Triplex assay kit (Genes2Life G2L-DeCoV19-MP, Guanajuato, Mexico). Identification of SARS-CoV-2 variants was also performed using the Master Mut Kit assay (Genes2Life G2LMUSC-14, Guanajuato, Mexico) as previously described [ 21 ]. The LaDEER eligibility criteria for RT-PCR testing of suspected COVID-19 patients has previously been published in greater detail [ 22 , 23 ].…”