WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of EnterovirusesA-Dtargeting the 5 ' UTR region

摘要

Aims The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to speciesA-Dtargeting 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 (R) Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regardingEnterovirus B,CandDwas determined to be 0 center dot 30 CCID(50)assay(-1)while the sensitivity forEnterovirus Awas 3 center dot 00 CCID(50)assay(-1). The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of EnterovirusesA-Dand 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 EVA-Dreference strains, on 20 EVBclinical isolates and on three non-enteroviral RNA viruses.