Centre : Central Potato Research Institute(CPRI)
Name of the Protocol
Development of ELISA based diagnosis for P. infestans
Nature : Novel
Crop / Pathogen : Potato/Phytophthora infestans
P. infestans cell wall based protein gene (392bp) has been isolated and cloned into pETSUMO expression vector. Positive clones were confirmed through colony PCR by using vector specific T7 promoter primer and revealed the 650bp fragment size and confirmed the integration of the gene. Protein was extracted and purified using G-Bioscience protein extraction kit. The purified protein was detected through SDS PAGE, which revealed the ~12KDa protein size and has been customised for developing polyclonal antibody. Polyclonal antibody of cell wall bound cellulose binding protein (CBD) of P. infestans was raised in rabbits, purified, analyzed by SDS PAGE (90%) and subjected to validation through DAC-ELISA. The recombinant CBD1 antiserum was evaluated by measuring its titre and efficacy in detecting P.infestans using DAC-ELISA. The expression of CBD1 was detected 2 fold over buffer control in mycelium, sporangium and zoospores as well as in infected leaf samples. Data indicated that the produced recombinant antiserum was efficient and accurate in determining negative and positive results in ELISA tests. Therefore, this antiserum is suitable for certification programs of potatoes due to its low cost, high specificity, feasibility and its endless supply from recombinant bacterial clones carrying the CBD genes for this oomycete.