1. A RT-PCR assay for the detection of coronaviruses from four genera.
- Author
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Xiu L, Binder RA, Alarja NA, Kochek K, Coleman KK, Than ST, Bailey ES, Bui VN, Toh TH, Erdman DD, and Gray GC
- Subjects
- Animals, Betacoronavirus genetics, Betacoronavirus isolation & purification, Bird Diseases virology, Birds, COVID-19, Coronavirus genetics, Coronavirus Infections virology, Genetic Variation, Humans, Pandemics, Pneumonia, Viral virology, Severe acute respiratory syndrome-related coronavirus genetics, Severe acute respiratory syndrome-related coronavirus isolation & purification, SARS-CoV-2, Swine, Swine Diseases virology, Bird Diseases diagnosis, Coronavirus isolation & purification, Coronavirus Infections diagnosis, Molecular Diagnostic Techniques methods, Pneumonia, Viral diagnosis, Reverse Transcriptase Polymerase Chain Reaction methods, Swine Diseases diagnosis
- Abstract
Background: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats., Objective: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily., Study Design and Results: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 10
2 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types., Conclusions: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily., Competing Interests: Declaration of Competing Interest The authors have declared that no competing financial interests exist., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
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