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Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.
J Virol Methods. 2018 08; 258:41-48.JV

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.

Authors+Show Affiliations

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.Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.Influenza virus Research Center, National Institute of Infectious Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.Eiken Chemical Co., Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan.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.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.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.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29763640

Citation

Shirato, Kazuya, et al. "Development of Fluorescent Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Using Quenching Probes for the Detection of the Middle East Respiratory Syndrome Coronavirus." Journal of Virological Methods, vol. 258, 2018, pp. 41-48.
Shirato K, Semba S, El-Kafrawy SA, et al. Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus. J Virol Methods. 2018;258:41-48.
Shirato, K., Semba, S., El-Kafrawy, S. A., Hassan, A. M., Tolah, A. M., Takayama, I., Kageyama, T., Notomi, T., Kamitani, W., Matsuyama, S., & Azhar, E. I. (2018). Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus. Journal of Virological Methods, 258, 41-48. https://doi.org/10.1016/j.jviromet.2018.05.006
Shirato K, et al. Development of Fluorescent Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Using Quenching Probes for the Detection of the Middle East Respiratory Syndrome Coronavirus. J Virol Methods. 2018;258:41-48. PubMed PMID: 29763640.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus. AU - Shirato,Kazuya, AU - Semba,Shohei, AU - El-Kafrawy,Sherif A, AU - Hassan,Ahmed M, AU - Tolah,Ahmed M, AU - Takayama,Ikuyo, AU - Kageyama,Tsutomu, AU - Notomi,Tsugunori, AU - Kamitani,Wataru, AU - Matsuyama,Shutoku, AU - Azhar,Esam Ibraheem, Y1 - 2018/05/12/ PY - 2018/02/23/received PY - 2018/04/09/revised PY - 2018/05/10/accepted PY - 2018/5/16/pubmed PY - 2018/10/3/medline PY - 2018/5/16/entrez KW - MERS coronavirus KW - Middle East respiratory syndrome KW - Quenching probe KW - RT-LAMP SP - 41 EP - 48 JF - Journal of virological methods JO - J Virol Methods VL - 258 N2 - 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. SN - 1879-0984 UR - https://www.unboundmedicine.com/medline/citation/29763640/Development_of_fluorescent_reverse_transcription_loop_mediated_isothermal_amplification__RT_LAMP__using_quenching_probes_for_the_detection_of_the_Middle_East_respiratory_syndrome_coronavirus_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(18)30105-8 DB - PRIME DP - Unbound Medicine ER -