title: Development of Dual TaqMan Based One-Step rRT-PCR Assay Panel for Rapid and Accurate Diagnostic Test of MERS-CoV: A Novel Human Coronavirus, Ahead of Hajj Pilgrimage.
abstract: 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.
  
  
  OBJECTIVE
  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).
  
  
  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.
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Mohammad Sadegh
authors.initials: MS
authors.lastname: Hashemzadeh
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Rahimeh
authors.initials: R
authors.lastname: Rasouli
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Bentolhoda
authors.initials: B
authors.lastname: Zahraei
authors.affiliation: Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Morteza
authors.initials: M
authors.lastname: Izadi
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Tat
authors.affiliation: Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Seyed Hassan
authors.initials: SH
authors.lastname: Saadat
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Najarasl
authors.affiliation: Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran.
authors.forename: Behzad
authors.initials: B
authors.lastname: Khansari Nejad
authors.affiliation: Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
authors.forename: Ruhollah
authors.initials: R
authors.lastname: Dorostkar
keywords: Diagnosis; Hajj Pilgrimage; MERS-CoV; ORF1b; Real-Time RT-PCR; upE