title: A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses.
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.
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. wanlv@126.com.|Medical Laboratory of Taizhou Fourth People's Hospital, Taizhou 225300, China. wanlv@126.com.
authors.forename: Zhenzhou
authors.initials: Z
authors.lastname: Wan
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. 13901433616@139.com.
authors.forename: Ya'nan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. kevin2003xzyz@163.com.
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: He
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. liujia02@sibs.ac.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. lanke@sibs.ac.cn.
authors.forename: Ke
authors.initials: K
authors.lastname: Lan
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. yhhu@ips.ac.cn.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China. zhangcy1999@ips.ac.cn.
authors.forename: Chiyu
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000208:Acute Disease; D017934:Coronavirus / Q000235:genetics / Q000302:isolation & purification*; D028941:Coronavirus 229E, Human / Q000235:genetics / Q000302:isolation & purification*; D058957:Coronavirus NL63, Human / Q000235:genetics / Q000302:isolation & purification*; D028962:Coronavirus OC43, Human / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000138:chemical synthesis / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003937:Diagnosis, Differential; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals / Q000737:chemistry; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D012367:RNA, Viral; C103389:SYTO 9
keywords: SYTO 9; human coronaviruses; melting curve; melting temperature (Tm); multiplex quantitative RT-PCR (RT-qPCR)