An improved DNA isolation method and PCR protocol for efficient detection of multicomponents of begomovirus in legumes

• Published in: Journal of virological methods 2008, Vol.147 (1), p.37-42
• Main Authors: Rouhibakhsh, A., Priya, J., Periasamy, M., Haq, Q.M.I., Malathi, V.G.
• Format: Article
• Language: English
• Subjects: Begomovirus; Begomovirus - genetics; Begomovirus - isolation & purification; Begomoviruses; Biological and medical sciences; DNA extraction; DNA, Viral - isolation & purification; Fabaceae - virology; Fundamental and applied biological sciences. Psychology; Legumes; Microbiology; Plant Diseases - virology; Polymerase Chain Reaction - methods; Techniques used in virology; Virology; Whitefly; Yellow mosaic
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.08.004

A relatively inexpensive protocol for the detection of genomic components of whitefly-transmitted begomoviruses in symptomatic legumes in the field is described. The method involves extraction with a modified CTAB buffer containing β-mercaptoethanol upto 5% and sodium chloride concentration from 1.4 to 2.0 M. Using this method PCR amplifiable DNA could be extracted from mature leaves of legume hosts rich in polyphenols, tannins and polysaccharides. The non-coding region and full-length DNA A, DNA B components of yellow mosaic viruses were consistently amplifiable from 97 samples, out of 136 tested in PCR reaction, employing primers specific for intergenic regions and full-length genome. The system is robust and the protocol is useful for the detection and identification of begomoviruses infecting grain legumes.