tetano
Editor, Senior Moderator
PLoS One
. 2022 Jan 13;17(1):e0261853.
doi: 10.1371/journal.pone.0261853. eCollection 2022.
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
Margaret G Mills[SUP] 1 [/SUP], Emily Bruce[SUP] 2 [/SUP], Meei-Li Huang[SUP] 1 [/SUP], Jessica W Crothers[SUP] 2 [/SUP], Ollivier Hyrien[SUP] 3 [/SUP], Christopher A L Oura[SUP] 4 [/SUP], Lemar Blake[SUP] 4 [/SUP], Arianne Brown Jordan[SUP] 5 [/SUP], Susan Hester[SUP] 6 [/SUP], Leah Wehmas[SUP] 6 [/SUP], Bernard Mari[SUP] 7 [/SUP], Pascal Barby[SUP] 7 [/SUP], Caroline Lacoux[SUP] 7 [/SUP], Julien Fassy[SUP] 7 [/SUP], Pablo Vial[SUP] 8 [/SUP], Cecilia Vial[SUP] 8 [/SUP], Jose R W Martinez[SUP] 8 [/SUP], Olusola Olalekan Oladipo[SUP] 9 [/SUP], Bitrus Inuwa[SUP] 10 [/SUP], Ismaila Shittu[SUP] 10 [/SUP], Clement A Meseko[SUP] 10 [/SUP], Roger Chammas[SUP] 11 [/SUP], Carlos Ferreira Santos[SUP] 12 [/SUP], Thiago José Dionísio[SUP] 12 [/SUP], Thais Francini Garbieri[SUP] 12 [/SUP], Viviane Aparecida Parisi[SUP] 12 [/SUP], Maria Cassia Mendes-Correa[SUP] 13 [/SUP], Anderson V de Paula[SUP] 13 [/SUP], Camila M Romano[SUP] 13 [/SUP], Luiz Gustavo Bentim Góes[SUP] 14 15 [/SUP], Paola Minoprio[SUP] 14 [/SUP], Angelica C Campos[SUP] 14 [/SUP], Marielton P Cunha[SUP] 14 [/SUP], Ana Paula P Vilela[SUP] 14 [/SUP], Tonney Nyirenda[SUP] 16 [/SUP], Rajhab Sawasawa Mkakosya[SUP] 16 [/SUP], Adamson S Muula[SUP] 17 [/SUP], Rebekah E Dumm[SUP] 18 [/SUP], Rebecca M Harris[SUP] 18 [/SUP], Constance A Mitchell[SUP] 19 [/SUP], Syril Pettit[SUP] 19 [/SUP], Jason Botten[SUP] 2 20 [/SUP], Keith R Jerome[SUP] 1 3 [/SUP]
Affiliations
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.
. 2022 Jan 13;17(1):e0261853.
doi: 10.1371/journal.pone.0261853. eCollection 2022.
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
Margaret G Mills[SUP] 1 [/SUP], Emily Bruce[SUP] 2 [/SUP], Meei-Li Huang[SUP] 1 [/SUP], Jessica W Crothers[SUP] 2 [/SUP], Ollivier Hyrien[SUP] 3 [/SUP], Christopher A L Oura[SUP] 4 [/SUP], Lemar Blake[SUP] 4 [/SUP], Arianne Brown Jordan[SUP] 5 [/SUP], Susan Hester[SUP] 6 [/SUP], Leah Wehmas[SUP] 6 [/SUP], Bernard Mari[SUP] 7 [/SUP], Pascal Barby[SUP] 7 [/SUP], Caroline Lacoux[SUP] 7 [/SUP], Julien Fassy[SUP] 7 [/SUP], Pablo Vial[SUP] 8 [/SUP], Cecilia Vial[SUP] 8 [/SUP], Jose R W Martinez[SUP] 8 [/SUP], Olusola Olalekan Oladipo[SUP] 9 [/SUP], Bitrus Inuwa[SUP] 10 [/SUP], Ismaila Shittu[SUP] 10 [/SUP], Clement A Meseko[SUP] 10 [/SUP], Roger Chammas[SUP] 11 [/SUP], Carlos Ferreira Santos[SUP] 12 [/SUP], Thiago José Dionísio[SUP] 12 [/SUP], Thais Francini Garbieri[SUP] 12 [/SUP], Viviane Aparecida Parisi[SUP] 12 [/SUP], Maria Cassia Mendes-Correa[SUP] 13 [/SUP], Anderson V de Paula[SUP] 13 [/SUP], Camila M Romano[SUP] 13 [/SUP], Luiz Gustavo Bentim Góes[SUP] 14 15 [/SUP], Paola Minoprio[SUP] 14 [/SUP], Angelica C Campos[SUP] 14 [/SUP], Marielton P Cunha[SUP] 14 [/SUP], Ana Paula P Vilela[SUP] 14 [/SUP], Tonney Nyirenda[SUP] 16 [/SUP], Rajhab Sawasawa Mkakosya[SUP] 16 [/SUP], Adamson S Muula[SUP] 17 [/SUP], Rebekah E Dumm[SUP] 18 [/SUP], Rebecca M Harris[SUP] 18 [/SUP], Constance A Mitchell[SUP] 19 [/SUP], Syril Pettit[SUP] 19 [/SUP], Jason Botten[SUP] 2 20 [/SUP], Keith R Jerome[SUP] 1 3 [/SUP]
Affiliations
- PMID: 35025926
- DOI: 10.1371/journal.pone.0261853
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.