BACKGROUND: Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses 
causing primarily respiratory disease in humans. A novel human coronavirus, 
subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was 
first reported in Saudi Arabia in September of 2012. With increasing numbers of 
infections and deaths from MERS-CoV, development of a rapid and reliable kit was 
crucial to prevent further spread of MERS-CoV.
OBJECTIVES: In this study, we present two real-time reverse-transcription 
polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive 
diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope 
gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening 
and final confirmation of MERS-CoV infection, as recommended by the world health 
organization (WHO).
MATERIALS AND METHODS: In this experimental study, acquiring patient samples was 
difficult; thus, according to WHO recommendations and standard protocols, we 
synthesized RNA sequences of upE and ORF1b genes as the template signatures and 
TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic 
genes for detection of MERS-CoV. In this research, we also inaugurated a 
cell-free system to transcribe these RNA sequences using the DNA templates 
synthesized.
RESULTS: The upE and ORF1b based one-step rRT-PCR assays were optimized by 
testing several times via different synthetic RNAs, and validation results were 
highly successful. The sensitivity obtained for upE was fewer than ten copies of 
RNA template per reaction and for ORF1b was 50 or fewer copies per reaction.
CONCLUSIONS: This study showed that the developed rRT-PCR assays are rapid, 
reliable, reproducible, specific, sensitive, and simple tools for detection of 
MERS-CoV. Finally, a kit consisting of two assay signatures and controls was 
assembled, which can be distributed to public health laboratories in Iran to 
support international MERS-CoV surveillance and public health response.