An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

PLoS One. 2022 Jan 13;17(1):e0261853. doi: 10.1371/journal.pone.0261853. eCollection 2022.

Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Grant support

MM:A grant from the Health and Environmental Sciences Institute to the University of Washington supported preparation and shipment of Project A and B samples to Propagate partner laboratories. HESI scientific staff also served as authors on the study but were not involved in direct sample preparation or data analysis. www.hesiglobal.org JB: An award from the University of Vermont (UVM) Office of the Vice President for Research supported work in the Botten lab at UVM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://www.uvm.edu/ovpr CV, PV: Fondecyt provided funding (grant 1201240) for the work of C. Vial and P. Vial. https://www.conicyt.cl/fondecyt/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. KJ: Grants from the National Institutes of Health National Institute of Allergy and Infectious Diseases (5R01AI129518 and UM1AI068635) and a gift from the MJ Murdock Charitable Trust helped to support the work of University of Washington and Fred Hutchinson Cancer Research Center researchers. https://www.niaid.nih.gov/ and https://murdocktrust.org/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.