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. 2016 Jun 21;18(11):e23874.
doi: 10.5812/ircmj.23874. eCollection 2016 Nov.

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

Mohammad Sadegh Hashemzadeh  1 , Rahimeh Rasouli  1 , Bentolhoda Zahraei  1 , Morteza Izadi  2 , Mahdi Tat  1 , Seyed Hassan Saadat  2 , Mohammad Najarasl  1 , Behzad Khansari Nejad  3 , Ruhollah Dorostkar  1
Affiliations

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

Mohammad Sadegh Hashemzadeh et al. Iran Red Crescent Med J. .

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.

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.

Keywords: Diagnosis; Hajj Pilgrimage; MERS-CoV; ORF1b; Real-Time RT-PCR; upE.

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Figures

Figure 1.
Figure 1.. Electrophoresis Analysis of upE and ORF1b Regions Amplified by 2% Gel-Based Traditional PCR
A, lane 1, 1 Kb DNA ladder (Fermentas); lane 2, upE amplicon with a length of ~90 bp; lane 3, negative control; B, Lane 1, 1 Kb DNA ladder (Fermentas); lane 2, ORF1b amplicon with a length of ~80 bp; lane 3, negative control; C, lanes 1 and 2, synthesized DNA template (plasmid) containing upE and ORF1b regions; lane 3, 1 Kb DNA Ladder (Fermentas).
Figure 2.
Figure 2.. Confirmatory Common Real-Time PCR Using the Corresponding Specific Probe and Primers (Forward and Reverse) of the upE Signature Assay for Several DNA Template Samples
Figure 3.
Figure 3.. Confirmatory Common Real-Time PCR Using the Corresponding Specific Probe and Primers (Forward and Reverse) of the ORF1b Signature Assay for Several DNA Template Samples
Figure 4.
Figure 4.. Confirmatory One-Step Real-Time RT-PCR Assay for One Standard RNA Sample Containing upE Signature Using Specific Probe and Primers
Figure 5.
Figure 5.. Confirmatory One-Step Real-Time RT-PCR Assay for One Standard RNA Sample Containing ORF1b Signature Using Specific Probe and Primers
Figure 6.
Figure 6.. Representative Amplification Plot of Developed rRT-PCR Assay, Showing Serial Dilutions of Standard RNAs for Evaluation of Analytical Sensitivity of Assays
See this image and copyright information in PMC

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