A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

Abstract

ABSTRACTEffective interventions are mandatory to control the transmission and spread of SARS-CoV-2, a highly contagious virus causing devastating effects worldwide. Cost-effective approaches are pivotal tools required to increase the detection rates and escalate further in massive surveillance programs, especially in countries with limited resources that most of the efforts have focused on symptomatic cases only. Here, we compared the performance of the RT-qPCR using an intercalating dye with the probe-based assay. Then, we tested and compared these two RT-qPCR chemistries in different pooling systems: after RNA extraction (post-RNA extraction) and before RNA extraction (pre-RNA extraction) optimizing by pool size and template volume. We evaluated these approaches in 610 clinical samples. Our results show that the dye-based technique has a high analytical sensitivity similar to the probe-based detection assay used worldwide. Further, this assay may also be applicable in testing by pool systems post-RNA extraction up to 20 samples. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 µL of RNA template. The low cost and the potential use in pre-RNA extraction pool systems, place of this assays as a valuable resource for scalable sampling to larger populations. Implementing a pool system for population sampling results in an important savings of laboratory resources and time, which are two key factors during an epidemic outbreak. Using the pooling approaches evaluated here, we are confident that it can be used as a valid alternative assay for the detection of SARS-CoV-2 in human samples.