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

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 spread of SARS-CoV-2 infection as well as aid in the development of informed policies for disease management. However, the current COVID-19 worldwide infection rates increased the demand for rapid and reliable screening of infection. We compared the performance of qRT-PCR in direct heat-inactivated (H), heat-inactivated and pelleted (HC) samples against RNA in a group of 74 subjects (44 positive and 30 negative). Then we compared the sensitivity of HC in a larger group of 196 COVID-19 positive samples. Our study suggests that HC samples show higher accuracy for SARS-CoV-2 detection PCR assay compared to direct H (89 % vs 83 % of the detection in RNA). The sensitivity of detection using direct samples varied depending on the sample transport and storage media as well as the viral loads (as measured by qRT-PCR Ct levels). Altogether, all the data suggest that purified RNA provides more accurate results, however, direct sample testing with 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.

Keywords: COVID-19; Direct sample qRT-PCR; Heat-Inactivation; Pelleting; SARS-CoV-2; Virus detection.

Fig. 1

Distribution of Ct values from COVID-19 patients nasopharyngeal swabs following qRT-PCR with standard RNA extraction, heat-inactivation (H), and heat-inactivation and pelleting (HC) methods. The limit of detection (40 Ct) is denoted with a dashed line. Samples with Ct values above this cutoff were considered negative for SARS-CoV-2 RNA. Samples are ordered by the purified RNA qRT-PCR Ct values. Only samples positive at least in one treatment method (RNA extraction, heat-inactivation, or heat-inactivation and pelleting) are presented. The summary of changes in Ct values is provided in Supplementary Table 1.

Fig. 2

Representative qRT-PCR amplification plots for the SARS-CoV-2 ORF1ab (FAM) and N (HEX) genes for a nasopharyngeal swab sample subjected to the standard RNA extraction, heat-inactivation (H), and heat-inactivation and pelleting (HC). Amplification plots show normalized value (ΔRn, linear scale) as a function of the qPCR cycle. The horizontal red line denotes the cycle threshold. (A) Plot shows the shift of Ct values towards higher values in direct samples. (B) Plot shows cases where Ct values for HC and H samples were smaller compared to the detection in extracted RNA samples (in 11.4 % of 44 positive samples) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).