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Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene

A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six...

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Autores principales: Wang, Yi, Wang, Yan, Xu, Huaqing, Dai, Hang, Meng, Shuang, Ye, Changyun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280119/
https://www.ncbi.nlm.nih.gov/pubmed/25549337
http://dx.doi.org/10.1371/journal.pone.0115868
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author Wang, Yi
Wang, Yan
Xu, Huaqing
Dai, Hang
Meng, Shuang
Ye, Changyun
author_facet Wang, Yi
Wang, Yan
Xu, Huaqing
Dai, Hang
Meng, Shuang
Ye, Changyun
author_sort Wang, Yi
collection PubMed
description A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.
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spelling pubmed-42801192015-01-07 Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene Wang, Yi Wang, Yan Xu, Huaqing Dai, Hang Meng, Shuang Ye, Changyun PLoS One Research Article A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories. Public Library of Science 2014-12-30 /pmc/articles/PMC4280119/ /pubmed/25549337 http://dx.doi.org/10.1371/journal.pone.0115868 Text en © 2014 Wang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wang, Yi
Wang, Yan
Xu, Huaqing
Dai, Hang
Meng, Shuang
Ye, Changyun
Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title_full Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title_fullStr Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title_full_unstemmed Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title_short Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene
title_sort rapid and sensitive detection of listeria ivanovii by loop-mediated isothermal amplification of the smcl gene
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280119/
https://www.ncbi.nlm.nih.gov/pubmed/25549337
http://dx.doi.org/10.1371/journal.pone.0115868
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