Open AccessAn 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
Author(s) -
Margaret G. Mills,
Emily A. Bruce,
MeeiLi Huang,
Jessica W Crothers,
Ollivier Hyrien,
Christopher Oura,
Lemar Blake,
Arianne Brown Jordan,
Susan Hester,
Leah C. Wehmas,
Bernard Mari,
Pascal Barby,
Caroline Lacoux,
Julien Fassy,
Pablo Vial,
Cecilia Vial,
Jose R W Martinez,
Olusola O. Oladipo,
Bitrus Inuwa,
Ismaila Shittu,
Clement Meseko,
Roger Chammas,
Carlos Santos,
Thiago José Dionísio,
Thais Francini Garbieri,
Viviane Aparecida Parisi,
Maria Cássia MendesCorrea,
Anderson Vincente de Paula,
Camila Malta Romano,
Luiz Gustavo Bentim Góes,
Paola Minóprio,
A. C. A. Campos,
Marielton dos Passos Cunha,
Ana Paula Pessoa Vilela,
Tonney S. Nyirenda,
Rajhab Sawasawa Mkakosya,
Adamson S Muula,
Rebekah E. Dumm,
Rebecca M. Harris,
Constance A. Mitchell,
Syril Pettit,
Jason Botten,
Keith R. Jerome
Publication year - 2022
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0261853
Subject(s) - rna extraction , reverse transcription polymerase chain reaction , real time polymerase chain reaction , polymerase chain reaction , covid-19 , coronavirus , reverse transcriptase , virology , medicine , computational biology , biology , rna , infectious disease (medical specialty) , pathology , disease , messenger rna , biochemistry , gene
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.