RCA-PCR: A ROBUST TECHNIQUE FOR THE DETECTION OF TOMATO LEAF CURL NEW DELHI VIRUS-POTATO AT ULTRA LOW VIRUS TITRE

Begomovirus is the largest genus of the family, Geminiviridae and comprises of whitefly-transmitted geminiviruses that infect dicotyledonous plants. During the last two decades these viruses have emerged as devastating pathogens, particularly in the tropics and subtropics, causing huge economic losses. Robust technique for detection of these viruses is a prerequisite for their effective management. The prevailing techniques for geminivirus detection include biological indexing, electron microscopy, antibody-based detection, including ELISA, nucleic acid hybridizations like dot blot and polymerase chain reaction using either specific or degenerate primers. However, fast evolving characteristics of the single-stranded viral genomes lead to failure of detection of these viruses through these techniques. To overcome these problems, many laboratories employ a rolling circle amplification (RCA) technique which in fact revolutionized the diagnosis of geminiviral infection. This technique has the advantage of amplifying any circular DNA without the initial knowledge of the sequence and it is better, easier and cheaper than polymerase chain reaction (PCR) or antibody based detection (Haible et al., 2006). RCA amplified viral DNAs can further be efficiently used for cloning and characterization of new geminiviruses. RCA combined with other techniques like RFLP and PCR also increases the sensitivity and specificity of the assay (Haible et al., 2006; Rocha et al., 2010).