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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
Journal
PLOS ONE
ISSN
1932-6203
Date Issued
2022
Author(s)
Margaret G. Mills
Emily Bruce
Meei-Li Huang
Jessica W. Crothers
Ollivier Hyrien
Christopher A. L. Oura
Lemar Blake
Arianne Brown Jordan
Susan Hester
Leah Wehmas
Bernard Mari
Pascal Barby
Caroline Lacoux
Julien Fassy
Jose R. W. Martinez
Olusola Olalekan Oladipo
Bitrus Inuwa
Ismaila Shittu
Clement A. Meseko
Roger Chammas
Carlos Ferreira Santos
Thiago José Dionísio
Thais Francini Garbieri
Viviane Aparecida Parisi
Maria Cassia Mendes-Correa
Anderson V. de Paula
Camila M. Romano
Luiz Gustavo Bentim Góes
Paola Minoprio
Angelica C. Campos
Marielton P. Cunha
Ana Paula P. Vilela
Tonney Nyirenda
Rajhab Sawasawa Mkakosya
Adamson S. Muula
Rebekah E. Dumm
Rebecca M. Harris
Constance A. Mitchell
Syril Pettit
Jason Botten
Keith R. Jerome
Type
Resource Types::text::journal::journal article
Abstract
<jats:p>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.</jats:p>
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