Tags

Type your tag names separated by a space and hit enter

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.
Iran Red Crescent Med J. 2016 Nov; 18(11):e23874.IR

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.

Authors+Show Affiliations

Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran.Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28191331

Citation

Hashemzadeh, Mohammad Sadegh, et al. "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." Iranian Red Crescent Medical Journal, vol. 18, no. 11, 2016, pp. e23874.
Hashemzadeh MS, Rasouli R, Zahraei B, et al. 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. Iran Red Crescent Med J. 2016;18(11):e23874.
Hashemzadeh, M. S., Rasouli, R., Zahraei, B., Izadi, M., Tat, M., Saadat, S. H., Najarasl, M., Khansari Nejad, B., & Dorostkar, R. (2016). 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. Iranian Red Crescent Medical Journal, 18(11), e23874. https://doi.org/10.5812/ircmj.23874
Hashemzadeh MS, et al. 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. Iran Red Crescent Med J. 2016;18(11):e23874. PubMed PMID: 28191331.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Hashemzadeh,Mohammad Sadegh, AU - Rasouli,Rahimeh, AU - Zahraei,Bentolhoda, AU - Izadi,Morteza, AU - Tat,Mahdi, AU - Saadat,Seyed Hassan, AU - Najarasl,Mohammad, AU - Khansari Nejad,Behzad, AU - Dorostkar,Ruhollah, Y1 - 2016/06/21/ PY - 2014/09/30/received PY - 2014/11/28/revised PY - 2015/04/05/accepted PY - 2017/2/14/entrez PY - 2017/2/14/pubmed PY - 2017/2/14/medline KW - Diagnosis KW - Hajj Pilgrimage KW - MERS-CoV KW - ORF1b KW - Real-Time RT-PCR KW - upE SP - e23874 EP - e23874 JF - Iranian Red Crescent medical journal JO - Iran Red Crescent Med J VL - 18 IS - 11 N2 - 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. SN - 2074-1804 UR - https://www.unboundmedicine.com/medline/citation/28191331/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_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28191331/ DB - PRIME DP - Unbound Medicine ER -