M Daskou1, T G Dimitriou1, D S Alexopoulou1, D Tsakogiannis1, G D Amoutzias2, D Mossialos1, Z Kyriakopoulou3, P Markoulatos1. 1. Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece. 2. Department of Biochemistry & Biotechnology, Bioinformatics Laboratory, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece. 3. Department of Microbiology Laboratory, General Department of Larissa, University of Thessaly, Biopolis, Larissa, Greece.
Abstract
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.
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.
Authors: Chao Tang; Sarah A Ahmed; Shuwen Deng; Lu Zhang; Jan Zoll; Abdullah M S Al-Hatmi; Jacques F Meis; Rameshwari Thakur; Yingqian Kang; G Sybren de Hoog Journal: Front Microbiol Date: 2022-08-23 Impact factor: 6.064