RNA was extracted from clinical specimens using either a QIAamp DSP Viral RNA Mini Kit (Catalog No. 61904, QIAGEN GmbH) or an automated MagNAPure 96 extraction instrument with a total nucleic acid isolation kit (Roche). The extraction was performed according to the manufacturers’ instructions and the extracted RNA was stored at −70°C.
MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR reaction mixture and 5 μL of template RNA). The thermocycling conditions included a reverse transcription reaction for 30 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A positive viral template control and a nontemplate control were included in each run. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified simultaneously as an internal control to monitor PCR inhibition. A positive result was identified by a well-defined exponential fluorescence curve that crossed the defined threshold at ≤35 cycles in both the upE and ORF1a assays.
MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR reaction mixture and 5 μL of template RNA). The thermocycling conditions included a reverse transcription reaction for 30 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A positive viral template control and a nontemplate control were included in each run. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified simultaneously as an internal control to monitor PCR inhibition. A positive result was identified by a well-defined exponential fluorescence curve that crossed the defined threshold at ≤35 cycles in both the upE and ORF1a assays.