A novel human coronavirus: Middle East respiratory syndrome human coronavirus

Geng, HeYuan; Tan, WenJie

To date, at least three real-time (RT-PCR) assays to screen and confirm MERS-CoV infection have been developed by Europe laboratories. The first RT-PCR screening assay (upE assay) was developed to target regions upstream of the E gene (27458-27550 nt), with sensitivity of 3.4 copies per reaction. Subsequently, a confirmatory assay targeting the ORF1b gene was developed, which does not overlap with the pan-coronavirus RT-PCR assays [18] . A third, confirmatory real-time RT-PCR assay, developed in Europe laboratories, targeted the ORF1a (1a assay) and its sensitivity was slightly lower than that of the upE assay. Furthermore, an immunofluorescence assay (IFA) to detect the antibody response was developed using convalescent patient serum that was biologically safe [19] . The upE assay used in combination with the 1a assay has been reported to provide a rigorously validated and highly sensitive result [20] . We have designed several primers and probes for real-time RT-PCR, modified using locked nucleic acid (LNA), to target the ORF1b gene with validated high sensitivity and no cross-reaction with HCoV-229E, -OC43, -NL63, -HKU1 and other respiratory pathogens [21] . The primers and probes designed in our laboratory and Europe laboratories are shown in Table 1 .