Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

Abstract

To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65˚C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10 3 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C T values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

“…Moreover, commonly used swab collection solutions may inhibit RT-PCR. Indeed, while several groups have demonstrated RNA amplification by direct addition of swab samples in the widely used viral transport medium (VTM), inhibition of RT-PCR by VTM typically leads to a significant delay in amplification [ 10 – 15 ]. A comparison of commercial master mixes found that the commonly used TaqPath master mix is particularly susceptible to inhibition by VTM [ 16 ].…”

“…An ideal swab collection solution would be widely available or cheaply made in any laboratory, allow for sensitive, direct detection of patient swabs, and not require specialized storage conditions. Proposed swab collection solutions include saline [ 18 ], PBS [ 14 ], TE [ 24 ], or simply distilled water [ 15 , 18 ]. Also, addition of proteinase K (PK) to UTM or saline was reported to improve detection of viral RNA by direct addition [ 19 , 25 ].…”

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

“…In other direct RT-PCR tests, saliva was diluted with a buffer and heated at 95 C for 30 min and then supplemented with Tween-20 [7] or heated at 65 C for 10 min [8]; an aliquot of the heat-treated, diluted sample was used for direct RT-PCR. The LOD for these tests, evaluated using saliva spiked with γ-irradiated SARS-CoV-2, was 1,000 copies of SARS-CoV-2 virus per ml of saliva [7], or 6,600 per ml of a viral suspension spiked with synthetic SARS-CoV-2 RNA [8]. Another direct RT-PCR test for detecting SARS-CoV-2 RNA used saliva treated with dithiothreitol (Sputasol) and a ribonuclease inhibitor [9].…”

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

“…Moreover, commonly used swab collection solutions may inhibit RT-PCR. Indeed, while several groups have demonstrated RNA amplification by direct addition of swab samples in the widely used viral transport medium (VTM), inhibition of RT-PCR by VTM typically leads to a significant delay in amplification [10][11][12][13][14][15]. A comparison of commercial master mixes found that the commonly used TaqPath master mix is particularly susceptible to inhibition by VTM [16].…”