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WarmStart colorimetric RT‐LAMP for the rapid, sensitive and specific detection of Enteroviruses A–D targeting the 5′UTR region
Author(s) -
Daskou M.,
Dimitriou T.G.,
Alexopoulou D.S.,
Tsakogiannis D.,
Amoutzias G.D.,
Mossialos D.,
Kyriakopoulou Z.,
Markoulatos P.
Publication year - 2021
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.14770
Subject(s) - amplicon , enterovirus , biology , microbiology and biotechnology , loop mediated isothermal amplification , virology , rna , complementary dna , enterovirus 71 , untranslated region , reverse transcriptase , polymerase chain reaction , dna , virus , gene , genetics
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 CCID 50 assay −1 while the sensitivity for Enterovirus A was 3·00 CCID 50 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.