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.

Synanthropic rodents as virus reservoirs and transmitters

“…Coronaviruses are enveloped, nonsegmented, positive, single-stranded RNA viruses associated with the structural N-phosphoprotein in a nucleocapsid with helical symmetry 112,113 . They are found in a wide variety of animals causing respiratory, enteric, hepatic, and neurological diseases of varying severity 114 . According to the International Committee on Taxonomy of Viruses, two subfamilies belong to the Coronaviridae family; Letovirinae, which has one subgenus, Milecovirus, found only in frogs and a sea hare thus far 115 , and Orthocoronavirinae, which is found in birds and mammals, and is divided into four genera due to the antigenic and genetic characteristics of the viruses 53,116 .…”

“…Betacoronaviruses are found mainly in mammals, such as humans, dogs, cats, pigs, bats, mice, rats, horses, and cattle 114,[118][119][120][121][122][123] , while Gama and Deltacoronaviruses infect mainly birds, with exceptions such as the white whale Gamacoronavirus (Delphinapterus leucas) 124 and the porcine Deltacoronavirus 125 .…”

Recent advances and perspectives of nucleic acid detection for coronavirus

“…Real-time reverse transcriptase-PCR (RT-PCR) detection is currently favored for the detection of coronavirus because of its advantages as a specific, and simple quantitative assay. Moreover, realtime RT-PCR is more sensitive than the conventional RT-PCR assay, which help much for the diagnosis in early infection [10,11]. Therefore, the real-time RT-PCR assay still is a predominant method to be applied for the detection of all kinds of coronaviruses [12,13], including SARS-CoV-2 [14].…”

Real-time RT-PCR in COVID-19 detection: issues affecting the results

“…Among nucleic acid tests, the polymerase chain reaction (PCR) method is considered as the 'gold standard' for the detection of some viruses and is characterized by rapid detection, high sensitivity, and specificity. As such, real-time reverse transcriptase-PCR (RT-PCR) is of great interest today for the detection of SARS-CoV -2 due to its benefits as a specific and simple qualitative assay [1][2][3]. Moreover, real-time RT-PCR has adequate sensitivity to help us much for diagnosing early infection.…”