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
Kazuya ShiratoEsam Ibraheem Azhar

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 detect...Continue Reading

Citations

Jul 26, 2019·Microbiology and Immunology·Kazuya Shirato
Jan 17, 2020·Journal of Medical Virology·Naru ZhangShibo Jiang
Sep 29, 2020·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·Ehsan ShabaniMohammad J Abdekhodaie
Aug 28, 2020·Frontiers in Microbiology·Lirong BaoJianxun Sun
Jul 24, 2020·International Journal of Molecular Sciences·Navid RabieeRajender S Varma
Sep 4, 2020·PeerJ·Tingting HuQingnan He
Aug 6, 2020·International Journal of Molecular Sciences·Franklin Wang-Ngai ChowSusanna Kar-Pui Lau
Oct 8, 2020·Molecular Biology Reports·Pranav Kumar Prabhakar, Jyoti Lakhanpal
Nov 22, 2020·Analytical and Bioanalytical Chemistry·Mercy R BenzigarGuozhen Liu
Dec 12, 2020·The Journal of Gene Medicine·Malihe MohamadianAbdolreza Esmaeilzadeh
Jan 9, 2021·Biophysical Chemistry·Chitrangada Das MukhopadhyayKeshav Rajarshi
Jul 28, 2020·Environment International·Isaac Dennis AmoahFaizal Bux
Feb 18, 2021·Talanta·Jiaqi BuShian Zhong
Nov 17, 2020·Frontiers in Public Health·José Adão Carvalho Nascimento JuniorMairim Russo Serafini
Feb 25, 2021·Microchemical Journal, Devoted to the Application of Microtechniques in All Branches of Science·Ahmad Mobed, Ebrahim Sepehri Shafigh
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May 1, 2021·Viruses·Shiori SanoMasayuki Saijo
Oct 17, 2021·Molecular Biotechnology·Mehrnaz EntesariFariba Rafiei
Dec 18, 2021·Seminars in Respiratory and Critical Care Medicine·Luna ColagrossiCarlo Federico Perno
Jan 15, 2022·Journal of Food Science·Ruibin XieAiliang Chen

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Datasets Mentioned

BETA
JX869059.2

Methods Mentioned

BETA
PCR

Software Mentioned

QProbe
PrimerExplorer
QProbe RT - LAMP
ESEQuant

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