Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

doi:10.1128/JCM.01224-15

Comparative Study

. 2015 Aug;53(8):2722-6.

doi: 10.1128/JCM.01224-15. Epub 2015 May 27.

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

Affiliations

¹ State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region, China Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China.
² Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China.
³ Department of Pathology, Hong Kong Sanatorium and Hospital, Hong Kong Special Administrative Region, China.
⁴ State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region, China Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China kyyuen@hku.hk.

PMID: 26019210
PMCID: PMC4508434
DOI: 10.1128/JCM.01224-15

Comparative Study

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

Jasper Fuk-Woo Chan et al. J Clin Microbiol. 2015 Aug.

. 2015 Aug;53(8):2722-6.

doi: 10.1128/JCM.01224-15. Epub 2015 May 27.

Authors

Affiliations

¹ State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region, China Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China.
² Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China.
³ Department of Pathology, Hong Kong Sanatorium and Hospital, Hong Kong Special Administrative Region, China.
⁴ State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong Special Administrative Region, China Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China kyyuen@hku.hk.

PMID: 26019210
PMCID: PMC4508434
DOI: 10.1128/JCM.01224-15

Abstract

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

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Figures

**FIG 1**
Schematic diagram of the MERS-CoV genome, with the leader sequence at the 5′-untranslated region enlarged to illustrate the abundance of the small RNA sequences. The percentages of mapped small RNA sequence reads at the leader sequence, ORF1a, S, and N gene regions are quantified and shown. Leader sequences of 70 to 72 nucleotides in length are also present in other human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1).

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References

1. Chan JF, To KK, Tse H, Jin DY, Yuen KY. 2013. Interspecies transmission and emergence of novel viruses: lessons from bats and birds. Trends Microbiol 21:544–555. doi:10.1016/j.tim.2013.05.005. - DOI - PMC - PubMed
1. Chan JF, To KK, Chen H, Yuen KY. 2015. Cross-species transmission and emergence of novel viruses from birds. Curr Opin Virol 10:63–69. doi:10.1016/j.coviro.2015.01.006. - DOI - PMC - PubMed
1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed
1. Cheng VC, Lau SK, Woo PC, Yuen KY. 2007. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 20:660–694. doi:10.1128/CMR.00023-07. - DOI - PMC - PubMed
1. Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY. 2012. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect 65:477–489. doi:10.1016/j.jinf.2012.10.002. - DOI - PMC - PubMed

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[1] Chan JF, To KK, Tse H, Jin DY, Yuen KY. 2013. Interspecies transmission and emergence of novel viruses: lessons from bats and birds. Trends Microbiol 21:544–555. doi:10.1016/j.tim.2013.05.005. - DOI - PMC - PubMed

[2] Chan JF, To KK, Tse H, Jin DY, Yuen KY. 2013. Interspecies transmission and emergence of novel viruses: lessons from bats and birds. Trends Microbiol 21:544–555. doi:10.1016/j.tim.2013.05.005. - DOI - PMC - PubMed

[3] Chan JF, To KK, Chen H, Yuen KY. 2015. Cross-species transmission and emergence of novel viruses from birds. Curr Opin Virol 10:63–69. doi:10.1016/j.coviro.2015.01.006. - DOI - PMC - PubMed

[4] Chan JF, To KK, Chen H, Yuen KY. 2015. Cross-species transmission and emergence of novel viruses from birds. Curr Opin Virol 10:63–69. doi:10.1016/j.coviro.2015.01.006. - DOI - PMC - PubMed

[5] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed

[6] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed

[7] Cheng VC, Lau SK, Woo PC, Yuen KY. 2007. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 20:660–694. doi:10.1128/CMR.00023-07. - DOI - PMC - PubMed

[8] Cheng VC, Lau SK, Woo PC, Yuen KY. 2007. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 20:660–694. doi:10.1128/CMR.00023-07. - DOI - PMC - PubMed

[9] Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY. 2012. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect 65:477–489. doi:10.1016/j.jinf.2012.10.002. - DOI - PMC - PubMed

[10] Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY. 2012. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect 65:477–489. doi:10.1016/j.jinf.2012.10.002. - DOI - PMC - PubMed

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Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

Affiliations

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

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