Real-time reverse transcription-PCR assay panel for Middle East respiratory syndrome coronavirus

Abstract

A new human coronavirus (CoV), subsequently named Middle East respiratory syndrome (MERS)-CoV, was first reported in Saudi Arabia in September 2012. In response, we developed two real-time reverse transcription-PCR (rRT-PCR) assays targeting the MERS-CoV nucleocapsid (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for the detection and confirmation of MERS-CoV infection. All assays detected ≤10 copies/reaction of quantified RNA transcripts, with a linear dynamic range of 8 log units and 1.3 × 10(-3) 50% tissue culture infective doses (TCID50)/ml of cultured MERS-CoV per reaction. All assays performed comparably with respiratory, serum, and stool specimens spiked with cultured virus. No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. In June 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an in vitro diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and a positive control was assembled and distributed to public health laboratories in the United States and internationally to support MERS-CoV surveillance and public health responses.

FIG 1

Plots of serial 10-fold dilutions ranging from 5 to 5 × 10⁷ copies/reaction for the N2 and N3 RNA transcripts and 10 to 10⁸ copies/reaction for the upE RNA transcripts analyzed by the MERS-CoV rRT-PCR assays. Plot inserts show calculated linear correlation coefficients (R²) and amplification efficiencies for each assay.

FIG 2

Algorithm for MERS-CoV rRT-PCR specimen testing and reporting of test results. For specimen screening, the N2 assay was combined with upE testing to potentially increase screening sensitivity and to reduce the possibility of false-negative results due to polymorphisms near the binding site of the oligonucleotide primers or probes. A positive test result by either or both assays requires confirmation with the N3 assay for reporting of a presumptive positive test result. VTC, viral template control; NTC, no-template control; RP, human RNase P gene.