External Quality Assessment of MERS-CoV Molecular Diagnostics During the 2015 Korean Outbreak

Seong, Moon-Woo; Lee, Seung Jun; Cho, Sung Im; Ko, Kyungphil; Kim, Mi-Na; Sung, Heungsub; Kim, Jae-Seok; Ahn, Ji Soo; Yu, Byung Su; Kim, Taek Soo; Kim, Eui Chong; Park, Sung Sup

For the first EQA phase, in vitro transcribed upE and ORF1a RNAs, provided by the University of Bonn Medical Center, Germany, were used at a concentration of 1,000 copies/μL. The test panel for the second EQA phase consisted of RNA extracts from three samples (Sample A, B, and C), two from MERS-CoV-positive patients (representing high positive and low positive levels), and one from a patient who was MERS-CoV-negative. All materials were assayed by using a rRT-PCR test for MERS-CoV RNA as previously described [3, 4] . Briefly, a 25-μL reaction was prepared with 5 μL of RNA, 1 μL of each primer, 0.5 μL of probe, 1 μL of enzyme mix, and 12.5 μL of 2 × RT-PCR buffer. Reverse transcription was done at 55°C for 20 min, and PCR cycling conditions were 94°C for 3 min followed by 45 cycles of 94°C for 15 sec, 58°C for 30 sec. The EQA samples were shipped frozen on dry ice by a specialist courier. The following information was collected from the participants: assay kit and platform used, cut-off CT value, and the results for each material.