Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR

Kim, Mi-Na; Ko, Young Jin; Seong, Moon-Woo; Kim, Jae-Seok; Shin, Bo-Moon; Sung, Heungsup

During July 6-10, 2015, each kit was validated by using the equipment recommended by each manufacturer (Table 1) . To determine analytical sensitivity, the limits of detection (LOD) with 95% probability values was determined by using upE and ORF1a RNA transcripts supplied by the Institute of Virology, University of Bonn Medical Centre [7] . The original concentration of both RNA transcripts was 1.0 × 10 5 copies/μL. These were diluted to six concentrations in 0.5-log steps from 100 to 0.3 copies/reaction, and kits were tested by using 5-8-μL samples of RNA eluates per reaction. For the Nanobiosys kit, which used 2.4-μL samples per reaction, a 0.5-log higher concentration was added for the LOD validation. Each concentration was tested by using 16 replicates, with the exception of PowerChek, for which 12 replicates were used. A probit regression analysis in R Studio (R Studio Inc.; https://www.rstudio.com/) was performed to determine the 95% cut-off values. The PowerChek, AccuPower, LightMix, and UltraFast LabChip kits used the primers and probes from the WHO-recommended rRT-PCR assay [7, To evaluate the analytical and clinical specificity of the kits, 28 respiratory virus-positive nasopharyngeal swabs were used to determine cross-reactions with human RNA or other respiratory viruses, including human coronaviruses. Using the Anyplex II RV16 kit (Seegene) with duplicate specimen preparations, these specimens were confirmed as positive for only single species of the following viruses: influenza virus A (n = 2), influenza virus B (n = 2), human parainfluenza virus 1 (n = 2), human parainfluenza virus 2 (n = 2), human parainfluenza virus 3 (n = 2), respiratory syncytial virus A (n = 2), respiratory syncytial virus B (n = 2), human adenovirus (n = 2), human bocavirus (n = 2), hu- man metapneumovirus (n = 2), human rhinovirus (n = 2), human coronavirus 229E (n = 2), human coronavirus OC43 (n = 2), and human coronavirus NL63 (n = 2).