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

International Journal of Molecular Sciences
Sumyya WaliullahMd Emran Ali

Abstract

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 fiel...Continue Reading

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Citations

Jan 29, 2021·Frontiers in Cellular and Infection Microbiology·Ganeshamoorthy Hariharan, Kandeeparoopan Prasannath

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Datasets Mentioned

BETA
M38183.1
AF325497
AY064391

Methods Mentioned

BETA
PCR
ELISA
nucleic acid hybridizations
electrophoresis

Software Mentioned

Primer Explorer
Genie explorer
Genie ® III
Geneious
[UNK]

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