Standardization of PCR protocol to check diversity among Pakistani isolates of cucumber mosaic Cucumovirus (CMV) infecting chilli pepper
Shomaila Iqbal, Hussain Shah, Muhammad Ashfaq and Ajmal Khan Kassi
Research was conducted in the year 2011-12 in NARC to standardize the protocol for the molecular characterization of CMV Pakistani isolates infecting chilli crop. Viral diseases are considered to be the major limiting factors in chili production. Cucumber mosaic virus is the type member of the Cucumovirus genus having isometric virus particles. It was detected through DAS-ELISA from the samples showing mosaic, leaf distortion, yellowing, and vein etching, stunting and yellowing like symptoms and also confirmed by RT-PCR. For evaluating the diversity different protocols were attempted for the standardization of RT-PCR against CMV. The coat protein (CP) gene of the CMV was amplified using RT-PCR with CP Gene-specific primers. Two steps protocol was finally followed with certain modifications; finally amplification product of an expected size [Australian Capsicum isolate (233 bp), Indian Caulicut Pepper isolate (232bp), Guangdong pepper isolate (218bp) and Indian longum pepper isolate (207 bp)] was obtained. Amplified product showed clear a band of an expected size whereas no satellite RNA was detected by using different sets of primers which shows that Pakistani isolate of CMV infecting chilli pepper belongs to 1b group.