SARS-CoV-2 detection by extraction-free qRT-PCR for massive and rapid COVID-19 diagnosis during a pandemic

Abstract

COVID-19 pandemic severely impacted the healthcare and economy on a global scale. It is widely recognized that mass testing is an efficient way to contain the infection spread as well as the development of informed policies for disease management. However, the current COVID-19 worldwide infection rates increased demand in the rapid and reliable screening of SARS-CoV-2 infection. We compared the performance of qRT-PCR in direct heat-inactivated, heat-inactivated/pelleted samples against RNA in a group of 74 subjects (44 positive and 30 negative). In addition, we compared the sensitivity of heat-inactivated/pelleted in another group of 196 COVID-19 positive samples. Our study suggests that swab sample heat-inactivation and pelleting show higher accuracy for SARS-CoV-2 detection PCR assay compared to heat-inactivation only (89% vs 83% of the detection in RNA). The accuracy of detection using direct samples varied depending on the sample transport and storage media as well as the viral titer. Our study suggests that purified RNA provides more accurate results, however, direct qRT-PCR may help to significantly increase testing capacity. Switching to the direct sample testing is justified if the number of tests is doubled at least.

A Simple and Cheap Method to Extract SARS-COV-2 Nucleic Acid from Nasopharyngeal Swab Without the Need Silica Filter Column

“…The second is the sample's purity. As a result of the use of direct samples in PCR reactions, the authors could only use 2l of VTM samples in a 20l PCR reaction to achieve the best results (10,11,14). To achieve a higher level of purity of samples, a simple and cost-benefit method was used to eliminate the inhibitors.…”

Implementation of an Extraction-Free COVID Real-Time PCR Workflow in a Pediatric Hospital Setting

“…Extraction-free real-time PCR assays eliminate the costly and time-intensive step of isolating nucleic acid from patient specimens. Variations of these assays have been published for the detection of SARS-CoV-2, testing varying temperatures for heat-inactivation ( 6–9 ) and buffers for stability ( 10 ). In the IDDL, we developed a modified version of a heat-inactivation protocol based on methods previously published by the Centers of Disease Control and Prevention ( 5 ), which has shown close correlation with traditional extraction procedures ( 7 , 8 , 11–13 ) and achieves full viral inactivation ( 14 ).…”