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.

Keywords: SYTO 9; human coronaviruses; melting curve; melting temperature (Tm); multiplex quantitative RT-PCR (RT-qPCR).

Figure 1

Melting curves of four HCoVs using the multiplex RT-qPCR assay. (A) The melting curves generated using first set of primers of each HCoV; (B) Improvement of Tm value of OC43 amplicon using GC-modified HCoV-OC43 primers; (C) The melting curves generated using the optimized primer sets with GC-modified HCoV-OC43 primers for four HCoVs.

Figure 2

Specificity of the multiplex RT-qPCR assay. (A) Amplification curves of 13 common respiratory viruses; (B) Melting curves of 13 common respiratory viruses. NTC, non-template control.

Figure 3

Sensitivity of the multiplex RT-qPCR assay using serially diluted RNA stocks. PDs, primer dimers; NTC, non-template control.