期刊
JOURNAL OF VIROLOGICAL METHODS
卷 174, 期 1-2, 页码 123-127出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2011.03.018
关键词
Sap; RT-PCR; Coleus; CbVd; Pipettor
资金
- Special Fund for Agro-scientific Research in the Public Interest [nyhyzx-200903004, 200903034]
- National Basic Research and Development Program of China (973 Program) [2009CB119200]
- National Natural Science Foundation of China [30771403, 30970134, 31071765]
- NSFC-JSPS China and Japan [30811140157]
- Key Project of the Fujian Provincial Department of Science and Technology [2007L2002]
- Grants-in-Aid for Scientific Research [21380029] Funding Source: KAKEN
A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.
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