Author/Authors :
Hashemzadeh، Mohammad Sadegh نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Rasouli، Rahimeh نويسنده Molecular Biology Unit, Pasteur Institute of Iran , , Zahraei، Bentolhoda نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Izadi، Morteza نويسنده , , Tat، Mahdi نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Saadat، Seyed-Hassan نويسنده , , Najarasl، Mohammad نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Khansari Nejad، Behzad نويسنده Department of Microbiology and Immunology, Arak University
of Medical Sciences, Arak, IR Iran , , Dorostkar، Ruhollah نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran ,
Abstract :
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. 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). 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. 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. 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.
