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.

Autor: Mills MG; Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America., Bruce E; Division of Immunobiology, Department of Medicine, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America., Huang ML; Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America., Crothers JW; Division of Immunobiology, Department of Medicine, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America., Hyrien O; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America., Oura CAL; School of Veterinary Medicine, University of the West Indies, St. Augustine, Trinidad and Tobago., Blake L; School of Veterinary Medicine, University of the West Indies, St. Augustine, Trinidad and Tobago., Brown Jordan A; Ministry of Health, Port of Spain, Trinidad and Tobago., Hester S; Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, United States of America., Wehmas L; Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, United States of America., Mari B; Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, FHU-OncoAge, Valbonne, France., Barby P; Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, FHU-OncoAge, Valbonne, France., Lacoux C; Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, FHU-OncoAge, Valbonne, France., Fassy J; Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, FHU-OncoAge, Valbonne, France., Vial P; Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Programa Hantavirus, Instituto de Ciencias e Innovación en Medicina, Santiago, Chile., Vial C; Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Programa Hantavirus, Instituto de Ciencias e Innovación en Medicina, Santiago, Chile., Martinez JRW; Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Programa Hantavirus, Instituto de Ciencias e Innovación en Medicina, Santiago, Chile., Oladipo OO; Biochemistry Division, National Veterinary Research Institute, Vom, Nigeria., Inuwa B; Infectious and Transboundary Animal Diseases, National Veterinary Research Institute, Vom, Nigeria., Shittu I; Infectious and Transboundary Animal Diseases, National Veterinary Research Institute, Vom, Nigeria., Meseko CA; Infectious and Transboundary Animal Diseases, National Veterinary Research Institute, Vom, Nigeria., Chammas R; Faculdade de Medicina da Universidade de São Paulo, Departamento de Radiologia e Oncologia, Centro de Investigação Translacional em Oncologia, Instituto do Cancer do Estado de São Paulo, São Paulo, Brazil., Santos CF; Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil., Dionísio TJ; Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil., Garbieri TF; Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil., Parisi VA; Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil., Mendes-Correa MC; Department of Infectious Diseases, Institute of Tropical Medicine of São Paulo, São Paulo, Brazil., de Paula AV; Department of Infectious Diseases, Institute of Tropical Medicine of São Paulo, São Paulo, Brazil., Romano CM; Department of Infectious Diseases, Institute of Tropical Medicine of São Paulo, São Paulo, Brazil., Góes LGB; Scientific Platform Pasteur-USP, Universidade de São Paulo, São Paulo, Brazil.; Department of Microbiology, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil., Minoprio P; Scientific Platform Pasteur-USP, Universidade de São Paulo, São Paulo, Brazil., Campos AC; Scientific Platform Pasteur-USP, Universidade de São Paulo, São Paulo, Brazil., Cunha MP; Scientific Platform Pasteur-USP, Universidade de São Paulo, São Paulo, Brazil., Vilela APP; Scientific Platform Pasteur-USP, Universidade de São Paulo, São Paulo, Brazil., Nyirenda T; Department of Pathology, College of Medicine, University of Malawi, Blantyre, Malawi., Mkakosya RS; Department of Pathology, College of Medicine, University of Malawi, Blantyre, Malawi., Muula AS; Department of Public Health, College of Medicine, University of Malawi, Blantyre, Malawi., Dumm RE; Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America., Harris RM; Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America., Mitchell CA; Health and Environmental Sciences Institute, Washington, DC, United States of America., Pettit S; Health and Environmental Sciences Institute, Washington, DC, United States of America., Botten J; Division of Immunobiology, Department of Medicine, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America.; Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America., Jerome KR; Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America.; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2022 Jan 13; Vol. 17 (1), pp. e0261853. Date of Electronic Publication: 2022 Jan 13 (Print Publication: 2022).
DOI: 10.1371/journal.pone.0261853
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje