Development of DNA based diagnostic systems for sugarcane pathogens : SRDC final report UQ024

Abstract

This project developed diagnostic assays for sugarcane pathogens using novel DNA detection technologies associated with the polymerase chain reaction (PCR). The bacterium Clavibacter xyli subsp. xyli (Cxx) causing ratoon stunting disease (RSD) was used as major model to compare assay platforms based on PCR-ELISA (Boehringer) and TaqManTM real time PCR (Applied Biosystems). TaqMan was more sensitive, robust, and subject to less interference than PCR-ELISA. Laboratory tests and field trials using cultured Cxx cells and xylem fluids from RSD-infected cane demonstrated that TaqMan could detect fewer than 10 bacterial cells reliably, and was >100x as sensitive as previous ELISA and phase contrast microscopy methods. Quantitative TaqMan assays appeared to be congruent with these existing methods. TaqMan assays were also developed for Fiji disease fijivirus (FDV) and sugarcane mosaic potyvirus (SCMV). BSES can readily access this advanced technology via the Real Time PCR Facility at the University of Queensland. Compared to alternative methods the TaqMan assays are inherently time-efficient, robust, highly sensitive, quantitative, and are especially well suited for rigorous quality control. These tests are recommended for ongoing evaluation for quarantine and other purposes by the Australian sugar industry. TaqMan is a robust generic technology and assays can readily be developed for further pathogens if the need arises.