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Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in...

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Autores principales: Waliullah, Sumyya, Ling, Kai-Shu, Cieniewicz, Elizabeth J., Oliver, Jonathan E., Ji, Pingsheng, Ali, Md Emran
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084362/
https://www.ncbi.nlm.nih.gov/pubmed/32143404
http://dx.doi.org/10.3390/ijms21051756
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author Waliullah, Sumyya
Ling, Kai-Shu
Cieniewicz, Elizabeth J.
Oliver, Jonathan E.
Ji, Pingsheng
Ali, Md Emran
author_facet Waliullah, Sumyya
Ling, Kai-Shu
Cieniewicz, Elizabeth J.
Oliver, Jonathan E.
Ji, Pingsheng
Ali, Md Emran
author_sort Waliullah, Sumyya
collection PubMed
description A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie(®) III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.
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spelling pubmed-70843622020-03-24 Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus Waliullah, Sumyya Ling, Kai-Shu Cieniewicz, Elizabeth J. Oliver, Jonathan E. Ji, Pingsheng Ali, Md Emran Int J Mol Sci Article A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie(®) III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics. MDPI 2020-03-04 /pmc/articles/PMC7084362/ /pubmed/32143404 http://dx.doi.org/10.3390/ijms21051756 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Waliullah, Sumyya
Ling, Kai-Shu
Cieniewicz, Elizabeth J.
Oliver, Jonathan E.
Ji, Pingsheng
Ali, Md Emran
Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title_full Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title_fullStr Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title_full_unstemmed Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title_short Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus
title_sort development of loop-mediated isothermal amplification assay for rapid detection of cucurbit leaf crumple virus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084362/
https://www.ncbi.nlm.nih.gov/pubmed/32143404
http://dx.doi.org/10.3390/ijms21051756
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