Location: Wheat, Sorghum and Forage Research
Project Number: 3042-22000-024-03-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2011
End Date: Jun 30, 2013
Specific objectives of this proposal are: 1) Optimization of TriMV antiserum produced against in vitro expressed coat protein (CP) in E. coli using various forms of ELISAs; 2) development of PCR and real-time reverse transcription (RT)-PCR detection methods for HPV; and 3) development of real-time RT-PCR method for quantitative detection of WSMV and TriMV in wheat curl mites.
During 2010-11 funding cycle, we produced polyclonal antiserum against coat protein (CP) of TriMV. The CP was over expressed in E. coli, which was isolated as soluble form and used as an antigen for antiserum production in rabbits (see progress report). In preliminary experiments, we found that this antiserum is specifically reacting with TriMV in DAS-ELISA. Different batches (bleeds) of TriMV antiserum are available in our lab, and these antisera will be optimized using different forms of ELISAs such as direct antigen coating and double antibody sandwich to choose a suitable ELISA method for sensitive and high-throughput detection of TriMV. We will perform partial genomic RNA sequencing from serologically distinct HPV isolates to obtain conserved stretches of sequences that can be used to design primers, and these primers will be used for PCR detection of HPV isolates from different regions of Nebraska. We will also utilize these conserved sequences in the genomes of HPV isolates to design primers and probe for real-time RT-PCR detection. The real-time PCR equipment is available in our lab. The real-time RT-PCR method will be optimized for quantitative detection of WSMV and TriMV in wheat curl mites. By using this method, we can quantify the number of virus particles present in mite vectors, which can be used to find the minimum number of virus particles required in mites to be able to transmit the virus by mite vectors.