Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.
Identifieur interne : 000902 ( PubMed/Corpus ); précédent : 000901; suivant : 000903Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.
Auteurs : Kazuya Shirato ; Shohei Semba ; Sherif A. El-Kafrawy ; Ahmed M. Hassan ; Ahmed M. Tolah ; Ikuyo Takayama ; Tsutomu Kageyama ; Tsugunori Notomi ; Wataru Kamitani ; Shutoku Matsuyama ; Esam Ibraheem AzharSource :
- Journal of virological methods [ 1879-0984 ] ; 2018.
English descriptors
- KwdEn :
- Animals, Camelus, Coronavirus Infections (diagnosis), Coronavirus Infections (veterinary), DNA Primers (genetics), Fluorescence, Humans, Middle East Respiratory Syndrome Coronavirus (genetics), Middle East Respiratory Syndrome Coronavirus (isolation & purification), Molecular Diagnostic Techniques (methods), Nucleic Acid Amplification Techniques (methods), Oligonucleotide Probes (genetics), Saudi Arabia, Sensitivity and Specificity.
- MESH :
- chemical , genetics : DNA Primers, Oligonucleotide Probes.
- geographic : Saudi Arabia.
- diagnosis : Coronavirus Infections.
- genetics : Middle East Respiratory Syndrome Coronavirus.
- isolation & purification : Middle East Respiratory Syndrome Coronavirus.
- methods : Molecular Diagnostic Techniques, Nucleic Acid Amplification Techniques.
- veterinary : Coronavirus Infections.
- Animals, Camelus, Fluorescence, Humans, Sensitivity and Specificity.
Abstract
Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.
DOI: 10.1016/j.jviromet.2018.05.006
PubMed: 29763640
Links to Exploration step
pubmed:29763640Le document en format XML
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Electronic address: shirato@nih.go.jp.</nlm:affiliation></affiliation></author><author><name sortKey="Semba, Shohei" sort="Semba, Shohei" uniqKey="Semba S" first="Shohei" last="Semba">Shohei Semba</name><affiliation><nlm:affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="El Kafrawy, Sherif A" sort="El Kafrawy, Sherif A" uniqKey="El Kafrawy S" first="Sherif A" last="El-Kafrawy">Sherif A. El-Kafrawy</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Hassan, Ahmed M" sort="Hassan, Ahmed M" uniqKey="Hassan A" first="Ahmed M" last="Hassan">Ahmed M. Hassan</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Tolah, Ahmed M" sort="Tolah, Ahmed M" uniqKey="Tolah A" first="Ahmed M" last="Tolah">Ahmed M. Tolah</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Takayama, Ikuyo" sort="Takayama, Ikuyo" uniqKey="Takayama I" first="Ikuyo" last="Takayama">Ikuyo Takayama</name><affiliation><nlm:affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Kageyama, Tsutomu" sort="Kageyama, Tsutomu" uniqKey="Kageyama T" first="Tsutomu" last="Kageyama">Tsutomu Kageyama</name><affiliation><nlm:affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Notomi, Tsugunori" sort="Notomi, Tsugunori" uniqKey="Notomi T" first="Tsugunori" last="Notomi">Tsugunori Notomi</name><affiliation><nlm:affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Kamitani, Wataru" sort="Kamitani, Wataru" uniqKey="Kamitani W" first="Wataru" last="Kamitani">Wataru Kamitani</name><affiliation><nlm:affiliation>Laboratory of Clinical Research on Infectious Diseases, Department of Pathogen Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><affiliation><nlm:affiliation>Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Azhar, Esam Ibraheem" sort="Azhar, Esam Ibraheem" uniqKey="Azhar E" first="Esam Ibraheem" last="Azhar">Esam Ibraheem Azhar</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2018">2018</date><idno type="RBID">pubmed:29763640</idno><idno type="pmid">29763640</idno><idno type="doi">10.1016/j.jviromet.2018.05.006</idno><idno type="wicri:Area/PubMed/Corpus">000902</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000902</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.</title><author><name sortKey="Shirato, Kazuya" sort="Shirato, Kazuya" uniqKey="Shirato K" first="Kazuya" last="Shirato">Kazuya Shirato</name><affiliation><nlm:affiliation>Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. Electronic address: shirato@nih.go.jp.</nlm:affiliation></affiliation></author><author><name sortKey="Semba, Shohei" sort="Semba, Shohei" uniqKey="Semba S" first="Shohei" last="Semba">Shohei Semba</name><affiliation><nlm:affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="El Kafrawy, Sherif A" sort="El Kafrawy, Sherif A" uniqKey="El Kafrawy S" first="Sherif A" last="El-Kafrawy">Sherif A. El-Kafrawy</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Hassan, Ahmed M" sort="Hassan, Ahmed M" uniqKey="Hassan A" first="Ahmed M" last="Hassan">Ahmed M. Hassan</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Tolah, Ahmed M" sort="Tolah, Ahmed M" uniqKey="Tolah A" first="Ahmed M" last="Tolah">Ahmed M. Tolah</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author><author><name sortKey="Takayama, Ikuyo" sort="Takayama, Ikuyo" uniqKey="Takayama I" first="Ikuyo" last="Takayama">Ikuyo Takayama</name><affiliation><nlm:affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Kageyama, Tsutomu" sort="Kageyama, Tsutomu" uniqKey="Kageyama T" first="Tsutomu" last="Kageyama">Tsutomu Kageyama</name><affiliation><nlm:affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Notomi, Tsugunori" sort="Notomi, Tsugunori" uniqKey="Notomi T" first="Tsugunori" last="Notomi">Tsugunori Notomi</name><affiliation><nlm:affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Kamitani, Wataru" sort="Kamitani, Wataru" uniqKey="Kamitani W" first="Wataru" last="Kamitani">Wataru Kamitani</name><affiliation><nlm:affiliation>Laboratory of Clinical Research on Infectious Diseases, Department of Pathogen Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><affiliation><nlm:affiliation>Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</nlm:affiliation></affiliation></author><author><name sortKey="Azhar, Esam Ibraheem" sort="Azhar, Esam Ibraheem" uniqKey="Azhar E" first="Esam Ibraheem" last="Azhar">Esam Ibraheem Azhar</name><affiliation><nlm:affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Journal of virological methods</title><idno type="eISSN">1879-0984</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Camelus</term><term>Coronavirus Infections (diagnosis)</term><term>Coronavirus Infections (veterinary)</term><term>DNA Primers (genetics)</term><term>Fluorescence</term><term>Humans</term><term>Middle East Respiratory Syndrome Coronavirus (genetics)</term><term>Middle East Respiratory Syndrome Coronavirus (isolation & purification)</term><term>Molecular Diagnostic Techniques (methods)</term><term>Nucleic Acid Amplification Techniques (methods)</term><term>Oligonucleotide Probes (genetics)</term><term>Saudi Arabia</term><term>Sensitivity and Specificity</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term><term>Oligonucleotide Probes</term></keywords><keywords scheme="MESH" type="geographic" xml:lang="en"><term>Saudi Arabia</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Coronavirus Infections</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Molecular Diagnostic Techniques</term><term>Nucleic Acid Amplification Techniques</term></keywords><keywords scheme="MESH" qualifier="veterinary" xml:lang="en"><term>Coronavirus Infections</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Camelus</term><term>Fluorescence</term><term>Humans</term><term>Sensitivity and Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29763640</PMID><DateCompleted><Year>2018</Year><Month>10</Month><Day>01</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>03</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1879-0984</ISSN><JournalIssue CitedMedium="Internet"><Volume>258</Volume><PubDate><Year>2018</Year><Month>08</Month></PubDate></JournalIssue><Title>Journal of virological methods</Title><ISOAbbreviation>J. Virol. Methods</ISOAbbreviation></Journal><ArticleTitle>Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.</ArticleTitle><Pagination><MedlinePgn>41-48</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S0166-0934(18)30105-8</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.jviromet.2018.05.006</ELocationID><Abstract><AbstractText>Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.</AbstractText><CopyrightInformation>Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Shirato</LastName><ForeName>Kazuya</ForeName><Initials>K</Initials><AffiliationInfo><Affiliation>Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. Electronic address: shirato@nih.go.jp.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Semba</LastName><ForeName>Shohei</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>El-Kafrawy</LastName><ForeName>Sherif A</ForeName><Initials>SA</Initials><AffiliationInfo><Affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hassan</LastName><ForeName>Ahmed M</ForeName><Initials>AM</Initials><AffiliationInfo><Affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tolah</LastName><ForeName>Ahmed M</ForeName><Initials>AM</Initials><AffiliationInfo><Affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Takayama</LastName><ForeName>Ikuyo</ForeName><Initials>I</Initials><AffiliationInfo><Affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kageyama</LastName><ForeName>Tsutomu</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Notomi</LastName><ForeName>Tsugunori</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kamitani</LastName><ForeName>Wataru</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>Laboratory of Clinical Research on Infectious Diseases, Department of Pathogen Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Matsuyama</LastName><ForeName>Shutoku</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Azhar</LastName><ForeName>Esam Ibraheem</ForeName><Initials>EI</Initials><AffiliationInfo><Affiliation>Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D023362">Evaluation Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>05</Month><Day>12</Day></ArticleDate></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>J Virol Methods</MedlineTA><NlmUniqueID>8005839</NlmUniqueID><ISSNLinking>0166-0934</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance 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