WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of Enteroviruses A–D targeting the 5′UTR region
Ημερομηνία
2021Γλώσσα
en
Λέξη-κλειδί
Επιτομή
Aims: The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A–D targeting the 5′ untranslated region (5′ UTR) of enteroviruses genome. Methods and Results: The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID50 assay−1 while the sensitivity for Enterovirus A was 3·00 CCID50 assay−1. The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A–D and validated on 20 clinical isolates. Conclusions: This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. Significance and Impact of the Study: We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5′ UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A–D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses. © 2020 The Society for Applied Microbiology
Collections
Related items
Showing items related by title, author, creator and subject.
-
Serum Neutralization Assay for the Determination of Antibody Levels Against Non-Polio Enterovirus Strains in Central and Western Greece
Fikatas A., Dimitriou T.G., Kyriakopoulou Z., Tsachouridou O., Gartzonika C., Levidiotou-Stefanou S., Amoutzias G.D., Markoulatos P. (2016)Mutations and recombination events have been identified in enteroviruses. Point mutations accumulate with a frequency of 6.3 × 10-4 per base pair per replication cycle affecting the fitness, the circulation, and the ... -
Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR
Fikatas A., Dimitriou T.G., Kyriakopoulou Z., Moschonas G.D., Amoutzias G.D., Mossialos D., Gartzonika C., Levidiotou-Stefanou S., Markoulatos P. (2017)In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the ... -
Development of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) that detects enteroviruses by targeting the highly conserved 5′-UTR region
Daskou M., Dimitriou T.G., Kouklamani-Giannouli G., Nikolaidis M., Mossialos D., Amoutzias G.D., Markoulatos P. (2020)Enteroviruses are positive sense single-stranded RNA viruses. Most infections from enteroviruses are asymptomatic and can circulate “silently”, increasing the risk of an outbreak. For preventing such outbreaks, a rapid, ...