OBJECTIVETo develop an one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses (HCoVs).

METHODSGenbank sequences of six human coronaviruses, including HCoV-NL63, HCoV-229E, SARS-CoV, HCoV-OC43, MERS-CoV, and HCoV-HKU1, were included as reference sequences. Primers were designed based on multiple alignment of reference sequences, targeting the conserved regions of each species of HcoV. Virus strains and nucleic acid preserved in our lab were used as template in developing this automatic-electrophoresis-based one-tube multiplex RT-PCR assay. Detection limits and reproducibility were also evaluated with these templates. Samples with infection of other respiratory viruses preserved in our lab were used to evaluate specificity of this assay. Finally, we tested this assay with 140 clinical samples that were validated by real-time PCR in parallel.

RESULTSThis automatic-electrophoresis-based multiplex RT-PCR assay was able to detect six human coronaviruses simultaneously. All positive samples in this study were detected with at least one specific fragment of anticipated length (195, 304, 332, 378, 415, 442 bp) . No fragment was detected in negative controls. Detection limits of 1.0×10(1-1.0)×10(2) copies/µl were achieved in tests of single virus. No cross reaction was observed with other respiratory viruses. This multiplex RT-PCR assay showed the same sensitivity and specificity to that of individual real-time RT-PCR validated with clinical samples. Both methods detected 28 positive samples (20%) .

CONCLUSIONSSix HCoVs can be detected in one tube by this novel multiplex RT-PCR assay with high sensitivity and specificity.