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PROJECT PROFILE. Title : “Referral Centre for Virus Testing of Tissue Culture Raised Plants” Duration : Five years Date of Start : 28 th September 2006
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PROJECT PROFILE Title : “Referral Centre for Virus Testing of Tissue Culture Raised Plants” Duration : Five years Date of Start : 28th September 2006 Date of Termination : 27th September 2011 Project Cost (Rs.) : 75.05 Lakhs Project Personnels Principal Investigator: R.K. Jain Co-Investigators : Padma Ramachandran/ V.K. Baranwal / Bikash Mandal Research Associates : Sudeep Bag / Surekha Agarwal
RESEARCH APPROACH • Confirmation of results on diagnosis of diagnostic test centers, whenever required • Development of diagnostic probes/kits for viruses • Development, standardization and validation of virus test protocols • Training of personnel of diagnostic centers • Maintenance of reference virus cultures and diagnostic reagents SERVICE APPROACH Major Objectives
SERVICE APPROACH Prepared write-up on Standard Operating Procedures (SOPs) for virus testing of tissue culture raised plants to be followed by Accrediated Test Laboratories (ATLs)
SERVICE APPROACH Imparted training to personnel from Accredited Test Laboratories and other Institutions ATLs Technical undergoing training
SERVICE APPROACH Supplied Diagnostic Reagent to various laboratories for virus detection
Service Approach Testing of plant samples for freedom from viruses
SERVICE APPROACH • Resource generation through testing of samples for freedom from viruses and other pathogens • Tissue culture industries • National Plant Quarantine Station Rs. 1,40,000/-
RESEARCH & DEVELOPMENT APPROACH Developed polyclonal antiserum to coat protein of PRSV using recombinant technology M 1 2 3 4 M 1 2 Amplification by RT-PCR Cloning in pGEMT Easy Vector ~850 bp ~850 bp Amplification of coat protein gene, Lane 1: helathy; Lane 2: infected sample PRSV infected leaf Mobilization of coat protein gene into expression vector (pMAL-C2X) M 1 2 3 4 5 6 Purification of expressed coat protein Production of polyclonal antiserum ~850 bp
Detection of Papaya ring spot virus (PRSV) in infected papaya and other hosts samples collected from different geographical locations by direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA)
RESEARCH & DEVELOPMENT APPROACH Developed polyclonal antiserum to coat protein of CTV using recombinant technology M 1 2 3 4 CTV infected material M 1 2 3 4 5 6 7 8 9 ~700 bp Cloning in pGEMT Easy Amplification by RT-PCR ~700 bp Mobilization of coat protein gene into expression vector (pMAL-C2X) M S IS FT W F4 F5 F8 F11MBP M S IS FT W F4 F5 F8 F11MBP Purification of expressed coat protein M 1 2 3 4 5 6 7 ~66 Kd ~66 Kd ~700 bp 50 Kd 50 Kd M : Marker; S: Soluble fraction; IS: Insoluble fraction; FT: Flowthrough; W: Wash; F4-11: eluted protein fraction Validation of Antiserum at IIHR, Bangalore and IARI, Regional Station, Kalimpong
Developed repository of diagnostic reagents for virus diagnosis
Acknowledgements • Department of Biotechnology DBT, New Delhi • Indian Agricultural Research Institute (IARI) , New Delhi • Research Associates- Sudeep Bag and Surekha Agarwal