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

UlS-ORF1a with the same RNA transcripts used to evaluate RealStar MERS-CoV (Altona Diagnostics), which is the only kit thus far approved for diagnosis [9] . When comparing the analytical sensitivities of assay systems, it is important to use consistent evaluation conditions; moreover, the quality of the source material is critical. It is often difficult to find traceable source materials for the molecular diagnosis of viral infections, as only a few international standards have been established to date [10] [11] [12] . As with RealStar MERS-CoV [9] , all target gene-binding sites for upE, ORF1a, and ORF1b used in the study kits were based on the oligonucleotide sequences of WHO-recommended primers and probes [3] , and the upE and ORF1a target sites were located within the RNA transcripts used in this study. Whole genome sequences of MERS-CoV isolates obtained during the 2015 Korean outbreak were determined by the Korea National Institute of Health and Seoul National University (gb|KT029139.1|) and the Guangdong Provincial Center for Disease Control and Prevention (gb|KT036372.1). Both sequences closely clustered with a strain isolated during the spring 2015 outbreak in Riyadh, Saudi Arabia (gb|KT026454.1|) [13] . Therefore, the primers and probes used in this study were most likely not affected by sequence variations in the MERS-CoV strains circulating in this outbreak. The RNA transcripts used to assess the analytical sensitivity in detecting upE and ORF1a [9] were not expected to have target mismatches with the primers and probes used in this study. Therefore, the LOD of all kits was analyzed by using identical source materials, and the data were comparable among various kits as well as with data from previous evaluations of RealStar MERS-CoV [9] . The LODs for upE of the kits assessed in this study indicated approximately 10-fold lower sensitivity than the previously reported 95% cut-off value of 5.3 copies/reaction for RealStar MERS-CoV [9] . The overall sensitivities for detecting upE of the kits evaluated herein were consistently lower than that of the RealStar MERS-CoV kit (Fig. 2) . Only the LOD for ORF1a using the PowerChek duplex kit was similar to that of the RealStar kit: 9.3 copies/reaction using the RealStar MERS-CoV kit in comparison with 6.9 copies/reaction using the PowerChek kit. Therefore, the lower sensitivities of the kits evaluated in comparison with that of RealStar MERS-CoV could be attributed to their performances rather than instability in source materials. In comparison with RealStar MERS-CoV [9] , all of the kits analyzed herein have room for improvement in their sensitivity, with the exception of ORF1a testing with the PowerChek kit. Unlike the other tested kits, the LightMix kit demonstrated a markedly trailing tendency to yield positive results down to much lower con-centrations than its LOD and an uncertain result to even a known-negative specimen (Fig. 1) . These findings suggest that the effort to increase sensitivity may result in more uncertainty and therefore negatively impact specificity. The previous study on the clinical sensitivity of RealStar MERS-CoV produced four discrepant results among 19 specimens, of which three specimens (oral, nasal, and urine specimens) showed high Ct values using the RealStar kit, but not using the comparison assay (and vice versa for one nasal specimen) [9] . This could be a result of RealStar MERS-CoV having a higher sensitivity or lower specificity than the other as