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
Identifieur interne : 000D74 ( Pmc/Corpus ); précédent : 000D73; suivant : 000D75Development 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
Auteurs : Mohammad Sadegh Hashemzadeh ; Rahimeh Rasouli ; Bentolhoda Zahraei ; Morteza Izadi ; Mahdi Tat ; Seyed Hassan Saadat ; Mohammad Najarasl ; Behzad Khansari Nejad ; Ruhollah DorostkarSource :
- Iranian Red Crescent Medical Journal [ 2074-1804 ] ; 2016.
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.
Url:
DOI: 10.5812/ircmj.23874
PubMed: 28191331
PubMed Central: 5292137
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">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</title><author><name sortKey="Hashemzadeh, Mohammad Sadegh" sort="Hashemzadeh, Mohammad Sadegh" uniqKey="Hashemzadeh M" first="Mohammad Sadegh" last="Hashemzadeh">Mohammad Sadegh Hashemzadeh</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Rasouli, Rahimeh" sort="Rasouli, Rahimeh" uniqKey="Rasouli R" first="Rahimeh" last="Rasouli">Rahimeh Rasouli</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Zahraei, Bentolhoda" sort="Zahraei, Bentolhoda" uniqKey="Zahraei B" first="Bentolhoda" last="Zahraei">Bentolhoda Zahraei</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Izadi, Morteza" sort="Izadi, Morteza" uniqKey="Izadi M" first="Morteza" last="Izadi">Morteza Izadi</name><affiliation><nlm:aff id="aff96433">Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Tat, Mahdi" sort="Tat, Mahdi" uniqKey="Tat M" first="Mahdi" last="Tat">Mahdi Tat</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Saadat, Seyed Hassan" sort="Saadat, Seyed Hassan" uniqKey="Saadat S" first="Seyed Hassan" last="Saadat">Seyed Hassan Saadat</name><affiliation><nlm:aff id="aff96433">Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Najarasl, Mohammad" sort="Najarasl, Mohammad" uniqKey="Najarasl M" first="Mohammad" last="Najarasl">Mohammad Najarasl</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Khansari Nejad, Behzad" sort="Khansari Nejad, Behzad" uniqKey="Khansari Nejad B" first="Behzad" last="Khansari Nejad">Behzad Khansari Nejad</name><affiliation><nlm:aff id="aff96434">Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Dorostkar, Ruhollah" sort="Dorostkar, Ruhollah" uniqKey="Dorostkar R" first="Ruhollah" last="Dorostkar">Ruhollah Dorostkar</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28191331</idno><idno type="pmc">5292137</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292137</idno><idno type="RBID">PMC:5292137</idno><idno type="doi">10.5812/ircmj.23874</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000D74</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D74</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">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</title><author><name sortKey="Hashemzadeh, Mohammad Sadegh" sort="Hashemzadeh, Mohammad Sadegh" uniqKey="Hashemzadeh M" first="Mohammad Sadegh" last="Hashemzadeh">Mohammad Sadegh Hashemzadeh</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Rasouli, Rahimeh" sort="Rasouli, Rahimeh" uniqKey="Rasouli R" first="Rahimeh" last="Rasouli">Rahimeh Rasouli</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Zahraei, Bentolhoda" sort="Zahraei, Bentolhoda" uniqKey="Zahraei B" first="Bentolhoda" last="Zahraei">Bentolhoda Zahraei</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Izadi, Morteza" sort="Izadi, Morteza" uniqKey="Izadi M" first="Morteza" last="Izadi">Morteza Izadi</name><affiliation><nlm:aff id="aff96433">Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Tat, Mahdi" sort="Tat, Mahdi" uniqKey="Tat M" first="Mahdi" last="Tat">Mahdi Tat</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Saadat, Seyed Hassan" sort="Saadat, Seyed Hassan" uniqKey="Saadat S" first="Seyed Hassan" last="Saadat">Seyed Hassan Saadat</name><affiliation><nlm:aff id="aff96433">Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Najarasl, Mohammad" sort="Najarasl, Mohammad" uniqKey="Najarasl M" first="Mohammad" last="Najarasl">Mohammad Najarasl</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Khansari Nejad, Behzad" sort="Khansari Nejad, Behzad" uniqKey="Khansari Nejad B" first="Behzad" last="Khansari Nejad">Behzad Khansari Nejad</name><affiliation><nlm:aff id="aff96434">Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran</nlm:aff></affiliation></author><author><name sortKey="Dorostkar, Ruhollah" sort="Dorostkar, Ruhollah" uniqKey="Dorostkar R" first="Ruhollah" last="Dorostkar">Ruhollah Dorostkar</name><affiliation><nlm:aff id="aff96432">Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</nlm:aff></affiliation></author></analytic><series><title level="j">Iranian Red Crescent Medical Journal</title><idno type="ISSN">2074-1804</idno><idno type="eISSN">2074-1812</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>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.</p></sec><sec><title>Objectives</title><p>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).</p></sec><sec><title>Materials and Methods</title><p>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.</p></sec><sec><title>Results</title><p>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.</p></sec><sec><title>Conclusions</title><p>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.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="King, Amq" uniqKey="King A">AMQ King</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, Am" uniqKey="Zaki A">AM Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author><author><name sortKey="Bestebroer, Tm" uniqKey="Bestebroer T">TM Bestebroer</name></author><author><name sortKey="Osterhaus, Ad" uniqKey="Osterhaus A">AD Osterhaus</name></author><author><name sortKey="Fouchier, Ra" uniqKey="Fouchier R">RA Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Memish, Za" uniqKey="Memish Z">ZA Memish</name></author><author><name 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Iran Red Crescent Med J</journal-id><journal-id journal-id-type="iso-abbrev">Iran Red Crescent Med J</journal-id><journal-id journal-id-type="doi">10.5812/ircmj</journal-id><journal-id journal-id-type="publisher-id">Kowsar</journal-id><journal-title-group><journal-title>Iranian Red Crescent Medical Journal</journal-title></journal-title-group><issn pub-type="ppub">2074-1804</issn><issn pub-type="epub">2074-1812</issn><publisher><publisher-name>Kowsar</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28191331</article-id><article-id pub-id-type="pmc">5292137</article-id><article-id pub-id-type="doi">10.5812/ircmj.23874</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-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</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Hashemzadeh</surname><given-names>Mohammad Sadegh</given-names></name><xref ref-type="aff" rid="aff96432">1</xref></contrib><contrib contrib-type="author"><name><surname>Rasouli</surname><given-names>Rahimeh</given-names></name><xref ref-type="aff" rid="aff96432">1</xref></contrib><contrib contrib-type="author"><name><surname>Zahraei</surname><given-names>Bentolhoda</given-names></name><xref ref-type="aff" rid="aff96432">1</xref></contrib><contrib contrib-type="author"><name><surname>Izadi</surname><given-names>Morteza</given-names></name><xref ref-type="aff" rid="aff96433">2</xref></contrib><contrib contrib-type="author"><name><surname>Tat</surname><given-names>Mahdi</given-names></name><xref ref-type="aff" rid="aff96432">1</xref></contrib><contrib contrib-type="author"><name><surname>Saadat</surname><given-names>Seyed Hassan</given-names></name><xref ref-type="aff" rid="aff96433">2</xref></contrib><contrib contrib-type="author"><name><surname>Najarasl</surname><given-names>Mohammad</given-names></name><xref ref-type="aff" rid="aff96432">1</xref></contrib><contrib contrib-type="author"><name><surname>Khansari Nejad</surname><given-names>Behzad</given-names></name><xref ref-type="aff" rid="aff96434">3</xref></contrib><contrib contrib-type="author"><name><surname>Dorostkar</surname><given-names>Ruhollah</given-names></name><xref ref-type="aff" rid="aff96432">1</xref><xref ref-type="corresp" rid="cor96435">*</xref></contrib></contrib-group><aff id="aff96432"><label>1</label>Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</aff><aff id="aff96433"><label>2</label>Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran</aff><aff id="aff96434"><label>3</label>Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran</aff><author-notes><corresp id="cor96435"><label>*</label>Corresponding Author: Ruhollah Dorostkar, Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran. Tel/Fax: +98-2188617711-16, E-mail: <email>R.dorost@yahoo.com</email></corresp></author-notes><pub-date pub-type="epub"><day>21</day><month>6</month><year>2016</year></pub-date><pub-date pub-type="collection"><month>11</month><year>2016</year></pub-date><volume>18</volume><issue>11</issue><elocation-id>e23874</elocation-id><history><date date-type="received"><day>30</day><month>9</month><year>2014</year></date><date date-type="rev-recd"><day>28</day><month>11</month><year>2014</year></date><date date-type="accepted"><day>05</day><month>4</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2016, Iranian Red Crescent Medical Journal</copyright-statement><copyright-year>2016</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/">http://creativecommons.org/licenses/by-nc/4.0/</ext-link>) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.</license-p></license></permissions><abstract><sec><title>Background</title><p>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.</p></sec><sec><title>Objectives</title><p>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).</p></sec><sec><title>Materials and Methods</title><p>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.</p></sec><sec><title>Results</title><p>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.</p></sec><sec><title>Conclusions</title><p>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.</p></sec></abstract><kwd-group kwd-group-type="author"><kwd>Hajj Pilgrimage</kwd><kwd>MERS-CoV</kwd><kwd>Diagnosis</kwd><kwd>Real-Time RT-PCR</kwd><kwd>upE</kwd><kwd>ORF1b</kwd></kwd-group></article-meta></front><body><sec id="sec185277"><title>1. Background</title><p>Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense ribonucleic acid (RNA) viruses (<xref rid="A23874R1" ref-type="bibr">1</xref>). They are widespread and can be found in many species of mice, horses, whales, birds, cats, dogs, pigs, and humans (<xref rid="A23874R1" ref-type="bibr">1</xref>). Development of the infection, can lead to additional complications, including respiratory tract disease and organ dysfunction, particularly renal failure and immune suppression, enteric, neurologic or hepatic diseases (<xref rid="A23874R2" ref-type="bibr">2</xref>, <xref rid="A23874R3" ref-type="bibr">3</xref>). The majority of patients have typical symptoms, such as fever with or without cough, and breathing difficulties (<xref rid="A23874R4" ref-type="bibr">4</xref>). Middle east respiratory syndrome coronavirus (MERS-CoV) is a new and unknown origin respiratory virus with genotypic and phenotypic diversity; thus, this virus can mutate, increasing its virulence and even causing tissue tropism. There is a high-frequency mortality rate around > 50% and median age of the majority of identified cases affected with this virus is 56 years. Very little is known about its behavior (<xref rid="A23874R5" ref-type="bibr">5</xref>, <xref rid="A23874R6" ref-type="bibr">6</xref>). Humans are known to maintain circulation of four different human coronaviruses (hCoVs) at a global population level. These are a part of the spectrum of agents that cause the common cold. The severe acute respiratory syndrome (SARS) CoV constitutes the fifth hCoV, which was in circulation for a limited time during 2002 and 2003, when a virus appeared in humans and caused an outbreak affecting at least 8,000 people. Symptoms matched the clinical picture of acute primary viral pneumonia. MERS-CoV, a novel coronavirus, was detected for the first time in September of 2012 in Saudi Arabia, an area heavily impacted by this virus at present. Between April and June 2013, 81 cases of infection by this hCoV were reported in Saudi Arabia. Of those, 49 patients died, so fatality rate is high (<xref rid="A23874R2" ref-type="bibr">2</xref>). This virus (MERS-CoV) causes severe acute respiratory infection in humans (<xref rid="A23874R7" ref-type="bibr">7</xref>). Subsequently MERS-CoV has been reported in other countries, including Tunisia, United Arab, Emirates, Italy, United Kingdom, Germany, France, and Qatar (<xref rid="A23874R2" ref-type="bibr">2</xref>, <xref rid="A23874R8" ref-type="bibr">8</xref>-<xref rid="A23874R11" ref-type="bibr">11</xref>). The first cases of MERS-CoV infections reported in Iran were two cases in Kerman, a city in southeast Iran. The virus killed one of them, a 53-year-old woman. With the appearance of new cases, in view of the risk of MERS-CoV transmission to humans (<xref rid="A23874R6" ref-type="bibr">6</xref>), and because of severe infections that have been observed among the elderly, there is considerable concern about this virus (<xref rid="A23874R5" ref-type="bibr">5</xref>). Although few cases have been reported annually (around 34 case as of 12 May 2013), the morbidity and mortality rate of this infection is alarming (<xref rid="A23874R7" ref-type="bibr">7</xref>). Unfortunately, at present, there are no specific treatments or effective drugs for this deadly disease, and no vaccine. In the absence of an effective treatment, the appropriate infection controls include rapid diagnostics and isolation of patients, useful strategies for preventing further transmission and spread of this infectious agent (<xref rid="A23874R5" ref-type="bibr">5</xref>). Currently, real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays are used for detection of MERS-CoV in respiratory, blood, and stool samples of patients. Real-time RT-PCR assay is highly sensitive and is able to detect viruses even in low copy numbers (<xref rid="A23874R12" ref-type="bibr">12</xref>, <xref rid="A23874R13" ref-type="bibr">13</xref>).</p></sec><sec id="sec185278"><title>2. Objectives</title><p>Our aims in this research were to present two 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 (according to world health organization (WHO) recommendations).</p></sec><sec id="sec185287"><title>3. Materials and Methods</title><sec id="sec185279"><title>3.1. Templates for Design of Assays</title><p>In this experimental study, the sequences, obtained from the National center for biotechnology information (NCBI; GenBank accession number: JX869059 for the Rotterdam virus isolate, termed hCoV-EMC), were synthesized (Eurofins Genomics, Germany) and served as templates for assay design. The DNA templates from upE and ORF1b of novel human coronavirus were amplified and confirmed using the corresponding real-time PCR specific probes and primers (forward and reverse) of each signature.</p></sec><sec id="sec185280"><title>3.2. Inauguration of a Cell-Free System for Transcription of Synthetic DNA Templates and Preparation of RNA Transcripts as Viral Templates</title><p>Target regions upE and ORF1b, synthesized by Eurofins Genomics (Germany) in the same vector (pGH cloning vector) were subcloned into the pSPT18 (Roche) expression vector using the HindIII restriction site (results not presented) and subsequently were transcribed into RNA for preparation of viral templates using the SP6/T7 Transcription kit (Roche) to synthesize both single-stranded RNAs. At all steps, contamination with RNase must be avoided through the usual diethylpyrocarbonate (DEPC) treatments, the use of disposable plasticware, wearing gloves, etc. After DNase I digestion, both RNA transcripts were purified using Roche High Pure Viral RNA Kit and quantified by ultraviolet (UV) light spectroscopy. No DNA content remained at this stage due to its removal, and results of common real-time PCR assays were negative (results not presented), whereas the results of real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays were positive.</p></sec><sec id="sec185281"><title>3.3. Real-Time RT-PCR Assay Panel Step 1: Suitable for Initial Screening of MERS-CoV Infection by Detection of Upstream Region of the Envelope (upE) Gene</title><p>The novel in-house one-step rRT-PCR assay was developed using the QuantiFast RT-PCR kit (Qiagen, Hilden, Germany) on Corbet (Rotor-Gene) 6000 (Qiagen, Germany) real-time PCR instrument. Each 25 µL reaction mixture contained 12.5 µL of 2 × Master Mix, 1 µL of reverse transcriptase/Taq DNA polymerase mixture, 0.4 mM of each dNTP and 3.2 mM magnesium sulfate, 5 μL of RNA, 400 nM concentrations of upE forward primer (GCAACGCGCGATTCAGTT) and upE reverse primer (GCCTCTACACGGGACCCATA), and 200 nM of upE probe (6-carboxyfluorescein [FAM]-CTCTTCACATAATCGCCCCGAGCTCG-6-carboxy-N, N, N, N’-tetramethylrhodamine [TAMRA]). Thermal cycling included 55°C for 20 minutes, followed by 95°C for 3 minutes and then 45 cycles of 95°C for 15 s and 58°C for 30 seconds. It should be mentioned that common one-step real-time RT-PCR kits formulated for application with probes should all provide satisfactory results with default reaction mix compositions as suggested by their manufacturers.</p></sec><sec id="sec185282"><title>3.4. Real-Time RT-PCR Assay Panel Step 2: Suitable for Final Confirmation of MERS-CoV Infection by Detection of Open Reading Frame (ORF) 1b Gene</title><p>This confirmatory assay had the same conditions as described above for the upE rRT-PCR, albeit the primer and probe sequences were different. This assay was performed with ORF1b forward primer (TTCGATGTTGAGGGTGCTCAT) and reverse primer (TCACACCAGTTGAAAATCCTAATTG), as well as its specific probe (6-carboxyfluorescein [FAM]-CCCGTAATGCATGTGGCACCAATGT-6-carboxy-N, N, N, N’ tetramethylrhodamine [TAMRA]). This target gene did not overlap with those of known pan-CoV assays (<xref rid="A23874R2" ref-type="bibr">2</xref>, <xref rid="A23874R6" ref-type="bibr">6</xref>, <xref rid="A23874R7" ref-type="bibr">7</xref>).</p></sec><sec id="sec185283"><title>3.5. In Vitro Synthesis of RNA Controls (Standard RNAs)</title><p>As noted above, two transcribed candidate signatures (target regions) upE and ORF1b, were used in both assays. Because of the difficulties in acquiring patient samples, according to WHO recommendation, these two RNA sequences were also synthesized (Eurofins Genomics, Germany) and used as RNA controls (standard RNAs) in optimization of the two assays and for preparation of serial dilutions for the evaluation of sensitivity. All RNA dilutions were prepared in RNase-free water.</p></sec><sec id="sec185286"><title>3.6. Evaluation of Analytical Sensitivity and Specificity of Assays</title><sec id="sec185284"><title>3.6.1. Analytical Sensitivity</title><p>A) Limit of detection - MERS-CoV upE gene: serial dilutions of quantified standard upE RNAs (10, 100, 1000, 10000) were prepared in 10 mM TE buffer and tested by upE assay signature in 25 replicates. The highest dilution of upE RNAs at which all replicates were positive was defined as the limit of detection (LoD) for the upE assay.</p><p>B) Limit of detection - MERS-CoV ORF1b gene: sensitivity evaluation was optimized by testing candidate probes and primers in the ORF1b signature assay with serial dilutions (5, 50, 500, 5000) of MERS-CoV quantified standard ORF1b RNAs in separate reactions and comparing them. Ten-fold serial dilutions of MERS-CoV ORF1b RNAs were prepared in buffer as above and tested in 25 replicates. The highest dilution of ORF1b RNAs at which all replicates were positive was defined as the limit of detection (LoD) for the ORF1b assay. A volume of 5 µL was used in two real-time RT-PCR assays of upE and ORF1b.</p></sec><sec id="sec185285"><title>3.6.2. Analytical Specificity</title><p>Reactivity with different MERS-CoV strains (in silico prediction): In addition to demonstrating reactivity of the rRT-PCR assay with the MERS-CoV strain Jordan-N3/NCV, primer/probe sequences were evaluated against an additional eight recently published genome sequences from seven patients collected from June 2012 to May 2013: GenBank accession numbers JX869059, KC776174, KC164505, KC667074, HPA Website (http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317138176202), KF192507, KF186564-KF186567. Primer/probe sequences for all signatures were 100% identical to all published virus strains (results not presented).</p></sec></sec></sec><sec id="sec185292"><title>4. Results</title><sec id="sec185288"><title>4.1. Confirmation of Synthesized DNA Template of upE and ORF1b</title><p>The DNA templates synthesized from upE and ORF1b of novel human coronavirus were amplified and confirmed using 2% gel-based traditional PCR (<xref ref-type="fig" rid="fig34285">Figure 1</xref>) and common real-time PCR with the corresponding specific probes and primers (forward and reverse) of each signature (<xref ref-type="fig" rid="fig34286">Figures 2</xref> and <xref ref-type="fig" rid="fig34287">3</xref>).</p><fig id="fig34285" orientation="portrait" position="float"><label>Figure 1.</label><caption><title>Electrophoresis Analysis of upE and ORF1b Regions Amplified by 2% Gel-Based Traditional PCR</title><p>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).</p></caption><graphic xlink:href="ircmj-18-11-23874-g001"></graphic></fig><fig id="fig34286" orientation="portrait" position="float"><label>Figure 2.</label><caption><title>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</title></caption><graphic xlink:href="ircmj-18-11-23874-i001"></graphic></fig><fig id="fig34287" orientation="portrait" position="float"><label>Figure 3.</label><caption><title>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</title></caption><graphic xlink:href="ircmj-18-11-23874-i002"></graphic></fig></sec><sec id="sec185289"><title>4.2. Real-Time RT-PCR Assay Panel Step 1: Suitable for Initial Screening of MERS-CoV Infection by Detection of Upstream Region of the Envelope (upE) RNA </title><p>This confirmatory one-step rRT-PCR assay was performed for synthetic standard RNA containing upE signature using specific probe and primers (<xref ref-type="fig" rid="fig34288">Figure 4</xref>). This test can initially screen for MERS-CoV infection bebecause it is a highly sensitive assay. The results of RNA transcripts (containing upE signature) were confirmatory, and were also similar to those of synthetic standard RNAs (results not presented here).</p><fig id="fig34288" orientation="portrait" position="float"><label>Figure 4.</label><caption><title>Confirmatory One-Step Real-Time RT-PCR Assay for One Standard RNA Sample Containing upE Signature Using Specific Probe and Primers</title></caption><graphic xlink:href="ircmj-18-11-23874-i003"></graphic></fig></sec><sec id="sec185290"><title>4.3. Real-Time RT-PCR Assay Panel Step 2: Suitable for Final Confirmation of MERS-CoV Infection by Detection of Open Reading Frame (ORF) 1b RNA</title><p>This confirmatory one-step rRT-PCR assay was performed for synthetic standard RNA containing ORF1b signature using specific probe and primers (<xref ref-type="fig" rid="fig34289">Figure 5</xref>). This test can conclusively detect MERS-CoV infection bebecause it is a highly specific assay. The results of RNA transcripts (containing ORF1b signature) were confirmatory, and were also similar to those of synthetic standard RNAs (results not presented here).</p><fig id="fig34289" orientation="portrait" position="float"><label>Figure 5.</label><caption><title>Confirmatory One-Step Real-Time RT-PCR Assay for One Standard RNA Sample Containing ORF1b Signature Using Specific Probe and Primers</title></caption><graphic xlink:href="ircmj-18-11-23874-i004"></graphic></fig></sec><sec id="sec185291"><title>4.4. Evaluation of Analytical Sensitivity of Assays</title><p>We prepared serial dilutions of standard RNAs for evaluation of analytical sensitivity of assays (<xref ref-type="fig" rid="fig34290">Figure 6</xref>). As shown in <xref ref-type="table" rid="tbl46905">Tables 1</xref> and <xref ref-type="table" rid="tbl46906">2</xref>, the stages of sensitivity (minimum detection limit) determination for each of these genes were done separately and the real-time RT-PCR assays showed different sensitivities for upE and ORF1b target regions.</p><fig id="fig34290" orientation="portrait" position="float"><label>Figure 6.</label><caption><title>Representative Amplification Plot of Developed rRT-PCR Assay, Showing Serial Dilutions of Standard RNAs for Evaluation of Analytical Sensitivity of Assays</title></caption><graphic xlink:href="ircmj-18-11-23874-i005"></graphic></fig><table-wrap id="tbl46905" orientation="portrait" position="float"><label>Table 1.</label><caption><title> MERS-CoV rRT-PCR Assay Limit of Detection for upE Gene<sup><xref ref-type="table-fn" rid="fn50045">a</xref></sup></title></caption><table frame="hsides" rules="groups"><thead><tr><th style="text-align: left;" rowspan="1" colspan="1">Serial Dilution (Copies/Reaction)</th><th rowspan="1" colspan="1">Positive Tests upE Region, %</th></tr></thead><tbody><tr><td rowspan="1" colspan="1"><bold>10</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr><tr><td rowspan="1" colspan="1"><bold>100</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr><tr><td rowspan="1" colspan="1"><bold>1000</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr><tr><td rowspan="1" colspan="1"><bold>10000</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr></tbody></table><table-wrap-foot><fn id="fn50045"><p><sup>a</sup>The highest dilution at which 100% of rRT-PCR replicates were positive is underlined.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl46906" orientation="portrait" position="float"><label>Table 2.</label><caption><title> MERS-CoV rRT-PCR Assay Limit of Detection for ORF1b Gene<sup><xref ref-type="table-fn" rid="fn50046">a</xref></sup></title></caption><table frame="hsides" rules="groups"><thead><tr><th style="text-align: left;" rowspan="1" colspan="1">Serial Dilution (Copies/Reaction)</th><th rowspan="1" colspan="1">Positive Tests upE Region, %</th></tr></thead><tbody><tr><td rowspan="1" colspan="1"><bold>5</bold></td><td style="text-align: center;" rowspan="1" colspan="1">10</td></tr><tr><td rowspan="1" colspan="1"><bold>50</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr><tr><td rowspan="1" colspan="1"><bold>500</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr><tr><td rowspan="1" colspan="1"><bold>5000</bold></td><td style="text-align: center;" rowspan="1" colspan="1">100</td></tr></tbody></table><table-wrap-foot><fn id="fn50046"><p><sup>a</sup>The highest dilution at which 100% of rRT-PCR replicates were positive is underlined.</p></fn></table-wrap-foot></table-wrap><p>A) Limit of detection (LoD) of the MERS-CoV upE gene: The serial dilutions containing 10, 100, 1000, and 10000 copies of standard RNAs per reaction were analyzed using the real-time RT-PCR assay. The highest dilution of upE RNAs at which all replicates were positive was defined as the limit of detection (LoD) for the upE assay. The minimum detection limit of the assay signature was fewer than ten copies of standard RNAs per reaction (<xref ref-type="table" rid="tbl46905">Table 1</xref>), as the higher dilutions were not evaluated.</p><p>B) Limit of detection (LoD) of the MERS-CoV ORF1b gene: The serial dilutions containing 5, 50, 500, and 5000 copies of standard RNAs per reaction were analyzed using the real-time RT-PCR assay. The highest dilution of ORF1b RNAs at which all replicates were positive was defined as the limit of detection (LoD) for the ORF1b assay. The minimum detection limit of the assay signature was 50 or fewer copies of standard RNAs per reaction (<xref ref-type="table" rid="tbl46906">Table 2</xref>), as the dilutions between 5 and 50 copies of standard RNAs per reaction were not evaluated.</p></sec></sec><sec id="sec185294"><title>5. Discussion</title><p>Since the human coronavirus (hCoV) termed SARS-CoV caused fatalities in Southeast Asia during 2002 and 2003, spread of another CoV, termed MERS-CoV, has caused considerable concern in the international community. As Saudi Arabia is the main center for the spread of MERS, it has a further importance in Islamic countries, where large numbers of people travel to Saudi Arabia for the Hajj pilgrimage. Annually, millions of world Muslims travel to Saudi Arabia in order to perform Hajj rituals and amassing of people can result in the spread of infection and disease transmission into different countries. Unfortunately, at present there is no specific treatment, such as an effective drug, and no vaccine for this deadly infection. In absence of an effective treatment, the appropriate infection controls include rapid diagnostics and isolation of patients, useful strategies for preventing further transmission and the spread of this infectious agent (<xref rid="A23874R5" ref-type="bibr">5</xref>). Development of a rapid and accurate diagnostic kit can facilitate early-stage detection of infected individuals with respiratory symptoms who have returned from the Hajj and are re-entering the country, preventing the spread of infection. The best methods for detection of MERS-CoV, recommended by WHO, are real-time RT-PCR assays, for upE gene as the initial screening of disease and for ORF1b (or ORF1a) gene as the final confirmation of MERS-CoV infection. It is notable that traditional (gel-based) RT-PCR methods require amplification in a thermo cycler (after cDNA preparation via RT process) and product analysis by electrophoresis gel, both of which are time-consuming and laborious (<xref rid="A23874R14" ref-type="bibr">14</xref>). By eliminating the need for post-amplification product processing, the real-time RT-PCR method enables shortened turn-around times for reporting results, which is critical for determining isolation for suspected case patients and contact tracing. Real-time RT-PCR assays are sensitive, rapid, and can be automated. They also have high reproducibility and since the need for post-PCR processing is eliminated, they can prevent carry-over contamination (<xref rid="A23874R14" ref-type="bibr">14</xref>). TaqMan probe-based real-time RT-PCR assays not only provide confidence in identification of target genes due to specific probing, but also reduce the risk of laboratory product contamination, because the amplification reaction and detection of PCR products are performed in a single tube (<xref rid="A23874R15" ref-type="bibr">15</xref>). Despite these advantages of rRT-PCR assays, these techniques are two-step. Step 1 is reverse transcription (RT) of RNA for cDNA preparation, and step 2 is the real-time PCR. In this study, we developed a novel one-step rRT-PCR assay, based on specific TaqMan probes, instead of traditional two-step rRT-PCR assays, enabling both steps to be accomplished in a single step. This increases the sensitivity of the assays and is affordable economically, in addition to the other advantages mentioned above.</p><p>Since, acquiring patient samples and the complete viral genome is difficult, according to WHO recommendations and standard protocols, we instead synthesized RNA sequences of upE and ORF1b genes as the template signatures, and the 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 RNAs using the synthesized DNA templates. As previously shown, the stages of sensitivity determination for each of these genes were done separately and the real-time RT-PCR assays showed different sensitivities for upE and ORF1b target regions. It is notable that the ORF1b assay has lower sensitivity than the upE assay.</p><sec id="sec185293"><title>5.1. Conclusion</title><p>This study showed that both the developed novel one-step rRT-PCR assays based on specific TaqMan probes are rapid, reliable, reproducible, specific, sensitive, and simple molecular methods for detection of MERS-CoV. Finally, the kit consisting of two assay signatures and controls was assembled, and it can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public health response.</p></sec></sec></body><back><ack><p>We thank Samaneh Zahiriyeganeh, Mohammad Reza Shafaati, Ehsan Zafari and Mojtaba Sharty for their contributions.</p></ack><fn-group><fn id="afn52897" fn-type="other"><p><bold>Authors’ Contribution:</bold>Ruhollah Dorostkar developed the original idea. Mohammad Sadegh Hashemzadeh wrote and prepared the manuscript. Rahimeh Rasouli, Bentolhoda Zahraei, Mahdi Tat, Mohammad Najarasl and Behzad Khansari Nejad contributed to the development of the protocols and operating activities. 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