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Int. J. Mol. Sci. 2016, 17(11), 1880; doi:10.3390/ijms17111880

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses

1
Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China
2
Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou 225300, China
These authors contributed equally to this study.
*
Authors to whom correspondence should be addressed.
Academic Editor: Stephen A. Bustin
Received: 21 July 2016 / Revised: 30 October 2016 / Accepted: 1 November 2016 / Published: 23 November 2016
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
View Full-Text   |   Download PDF [1694 KB, uploaded 23 November 2016]   |  

Abstract

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 102 copies for HCoV-OC43, and 3 × 101 copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples. View Full-Text
Keywords: human coronaviruses; melting curve; multiplex quantitative RT-PCR (RT-qPCR); SYTO 9; melting temperature (Tm) human coronaviruses; melting curve; multiplex quantitative RT-PCR (RT-qPCR); SYTO 9; melting temperature (Tm)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Wan, Z.; Zhang, Y.; He, Z.; Liu, J.; Lan, K.; Hu, Y.; Zhang, C. A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses. Int. J. Mol. Sci. 2016, 17, 1880.

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