Evaluation and Clinical Validation of Two Field-Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome-Coronavirus

Abstract

Middle East respiratory syndrome (MERS) is an emerging zoonotic viral respiratory disease that was first identified in Saudi Arabia in 2012. In 2015, the largest MERS outbreak outside of the Middle East region occurred in the Republic of Korea. The rapid nosocomial transmission of MERS-coronavirus (MERS-CoV) in Korean health care settings highlighted the importance and urgent need for a rapid and reliable on-site diagnostic assay to implement effective control and preventive measures. Here, the evaluation and validation of two newly developed reverse transcription-insulated isothermal PCR (RT-iiPCR) methods targeting the ORF1a and upE genes of MERS-CoV are described. Compared with World Health Organization-recommended singleplex real-time quantitative RT-PCR (RT-qPCR) assays, both RT-iiPCR assays had comparable analytical sensitivity for the detection of MERS-CoV RNA in tissue culture fluid and in sputum samples spiked with infectious virus. Furthermore, clinical evaluation was performed with sputum samples collected from subjects with acute and chronic respiratory illnesses, including patients infected with MERS-CoV. The overall agreement values between the two RT-iiPCR assays and the reference RT-qPCR assays were 98.06% (95% CI, 94.43%-100%; κ = 0.96) and 99.03% (95% CI, 95.88%-100%; κ = 0.99) for ORF1a and upE assays, respectively. The ORF1a and upE MERS-CoV RT-iiPCR assays coupled with a field-deployable system provide a platform for a highly sensitive and specific on-site tool for diagnosis of MERS-CoV infections.

Figure 1

POCKIT system workflow for point-of-need detection of Middle East respiratory syndrome–coronavirus (MERS-CoV) RNA. This system includes a compact automatic nucleic acid (NA) extraction device (taco mini) and a portable PCR device (POCKIT). After sample collection, nucleic acids were extracted using a preloaded extraction plate in approximately 30 minutes, and subsequently, the lyophilized reverse transcription-insulated isothermal PCR reaction was reconstituted and nucleic acids were added. The mixture was transferred to an R-tube and tested in a POCKIT device. TaqMan probe hydrolysis–based amplification signals were automatically detected, processed, and interpreted, providing qualitative results on the display screen in 60 minutes. Asterisk indicates sample collection method used in this study.