Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 213, Issue -, Pages 93-97Publisher
ELSEVIER
DOI: 10.1016/j.jviromet.2014.11.013
Keywords
Tomato chlorosis virus; RT-LAMP; RT-PCR; Detection; Isothermal amplification
Funding
- Special Fund for Agro-scientific Research in the Public Interest [201003065]
- Key technical project of Shandong Yan-cao Co. Ltd [KN214-201211]
- Provincial Natural Science Foundation of Shandong [ZR2012CM032]
- National Natural Science Foundation of China, NSFC [30871620]
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A betaine-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimised for detecting tomato chlorosis virus (ToCV), one of the most important viruses that infect tomato crops worldwide. A set of four specific primers was designed against the RNA-dependent RNA polymerase (RdRp) gene. The betaine-free RT-LAMP procedure could be completed within 40 min under isothermal conditions at 60 degrees C without a thermal cycler, and no cross-reactivity was seen with other tomato viral pathogens. Sensitivity analysis showed that RT-LAMP could detect viral dilutions up to 2.0 x 10(-7) ng, which is 100-times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR). In addition, naked-eye observation after staining in-tube RT-LAMP products with SYBR Green I facilitated detection of ToCV by avoiding the requirement for ethidium staining following gel electrophoresis. These results suggest that ToCV RT-LAMP is a rapid, sensitive, and affordable diagnostic tool that is more suitable than RT-PCR for the detection and surveillance of ToCV in field samples. (C) 2014 Elsevier B.V. All rights reserved.
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