Published April 30, 2020 | Version v1
Journal article Open

A RT-PCR assay for the detection of coronaviruses from four genera

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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 × 102 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

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References

  • J.F. Chan, S.K. Lau, K.K. To, V.C. Cheng, P.C. Woo, K.Y. Yuen, Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARSlike disease, Clin. Microbiol. Rev. 28 (2015) 465–522
  • Forni D., Cagliani R., Clerici M., Sironi M. Molecular evolution of human coronavirus genomes. Trends Microbiol. 2017;25:35–48.
  • ICTV: https://talk.ictvonline.org/ictv-reports/ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/222/coronaviridae (Accessed on April 9, 2020).
  • Guarner J. Three emerging coronaviruses in two decades. Am. J. Clin. Pathol. 2020;153:420–421
  • Wang Q., Vlasova A.N., Kenney S.P., Saif L.J. Emerging and re-emerging coronaviruses in pigs. Curr. Opin. Virol. 2019;34:39–49.
  • Zhou P., Fan H., Lan T., Yang X.L., Shi W.F., Zhang W. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature. 2018;556:255–258.
  • FindDx: https://www.finddx.org/covid-19/pipeline/?section=immunoassays#diag_tab (Accessed on April 9, 2020).
  • Toh T.H., Hii K.C., Fieldhouse J.K., Ting J., Berita A., Nguyen T.T. High prevalence of viral infections among hospitalized pneumonia patients in equatorial Sarawak. Malaysia. Open Forum Infect Dis. 2019;6 ofz074.
  • Bui V.N., Nguyen T.T., Nguyen-Viet H., Bui A.N., McCallion K.A., Lee H.S. Bioaerosol sampling to detect avian influenza virus in Hanoi's largest live poultry market. Clin. Infect. Dis. 2019;68:972–975.
  • Bailey ES, Borkenhagen LK, Choi JY, Greer AE, Gray GC. A Feasibility Study of Conducting Surveillance for Swine Pathogens in Swine Slurry in North Carolina Swine Farms. Under journal review.
  • CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2 (Accessed on April 18, 2020).
  • Hudu S.A., Alshrari A.S., Syahida A., Sekawi Z. Cell culture, technology: enhancing the culture of diagnosing human diseases. J. Clin. Diagn. Res. 2016;10:DE01–5.
  • Meyer B., Drosten C., Muller M.A. Serological assays for emerging coronaviruses: challenges and pitfalls. Virus Res. 2014;194:175–183.
  • Doan T., Pinsky B.A. Current and future molecular diagnostics for ocular infectious diseases. Curr. Opin. Ophthalmol. 2016;27:561–567.
  • Xiu L., Zhang C., Wu Z., Peng J. Establishment and application of a universal coronavirus screening method using MALDI-TOF mass spectrometry. Front. Microbiol. 2017;8:1510.
  • Corman V.M., Muth D., Niemeyer D., Drosten C. Hosts and sources of endemic human coronaviruses. Adv. Virus Res. 2018;100:163–188.
  • Corman V.M., Baldwin H.J., Tateno A.F., Zerbinati R.M., Annan A., Owusu M. Evidence for an ancestral association of human coronavirus 229E with bats. J. Virol. 2015;89:11858–11870.
  • Tao Y., Shi M., Chommanard C., Queen K., Zhang J., Markotter W. Surveillance of bat coronaviruses in Kenya identifies relatives of human coronaviruses NL63 and 229E and their recombination history. J. Virol. 2017;91
  • Huynh J., Li S., Yount B., Smith A., Sturges L., Olsen J.C. Evidence supporting a zoonotic origin of human coronavirus strain NL63. J. Virol. 2012;86:12816–12825.
  • Banerjee A., Kulcsar K., Misra V., Frieman M., Mossman K. Bats and coronaviruses. Viruses. 2019;11
  • Boley P.A., Alhamo M.A., Lossie G., Yadav K.K., Vasquez-Lee M., Saif L.J. Porcine deltacoronavirus infection and transmission in poultry, United States. Emerg Infect Dis. 2020;26:255–265.
  • Li W., Hulswit R.J.G., Kenney S.P., Widjaja I., Jung K., Alhamo M.A. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility. Proc Natl Acad Sci U S A. 2018;115:E5135–E5143.