1.13.1
DEVELOPMENT OF RT-PCR-ELISA AND TAQMANTM ASSAY FOR FIJI DISEASE VIRUS IN SUGARCANE AND ITS PLANTHOPPER VECTOR

L PICKERING1, RJ DIETZGEN2, J HENDERSON1, GR SMITH3, PJ WHITTLE3 and DJ MACLEAN1

1Co-operative Research Centre for Tropical Plant Pathology, Department of Biochemistry, University of Queensland, Qld 4072, Australia; 2Queensland Agricultural Biotechnology Centre, St Lucia, Qld 4072, Australia; 3Bureau of Sugar Experiment Stations, 20 Meiers Rd, Indooroopilly, Qld 4068, Australia

Background and objectives
Fiji disease virus (FDV) is a reovirus with a dsRNA genome, and is an important pathogen of major concern to the Australian sugarcane quarantine services. The first definitive symptoms observed in infected cane are raised, whitish galls on the undersurface of the leaf laminae. However, FDV is known to have a long latent period and the disease symptoms may not appear until after regrowth from the rootstock, many months after the initial infection [1]. The virus is transmitted by the delphacid planthopper Perkinsiella saccharicida. The virus is acquired in the first three instars of the insect life cycle and replicates persistently within the vector. However, it is not known if the virus is transmitted transovarially from parent to progeny within the vector. The aim was to develop highly sensitive and specific assay systems for the detection of FDV using RT-PCR-based technologies that avoid gel detection. We have developed suitable RT-PCR-ELISA and TaqManTM assays for the detection of FDV in sugarcane leaf tissue and the planthopper vector. The development of these assays should greatly assist the Australian sugar industry to index local and imported sugarcane germplasm.

Materials and methods
Both the RT-PCR-ELISA and TaqManTM detection assays confer dual specificity using both RT-PCR primers and internal hybridization probes. The target sequence for both assays was specific to one of the 1.7-kbp fragments from the segmented dsRNA genome of FDV. The RT-PCR-ELISA assay system, supplied by Boehringer Mannheim, incorporates digoxigenin (DIG) into the product during PCR. A 450-bp product amplified by RT-PCR primers FDV-7F and FDV-7R is detected using the biotinylated hybridization probe FDV-Bio7. The fluorescence-based TaqManTM system developed by Perkin-Elmer was also adapted to FDV and uses FDV-727F and FDV-Tq-7R RT-PCR primers to amplify a 200-bp fragment. The ABI Prism Model 7700 Sequence Detection System is used to detect the amplified product using the dye-labelled probe FDV-FAM-7, and records the accumulation of fluorescence in real time during successive PCR cycles [2].

Results and conclusions
Using purified FDV preparations, the TaqManTM assay was shown to be quantitative over seven orders of magnitude, with a detection limit of <10 FDV genomes/reaction. This is compared to the detection limit of 104 genomes/reaction for RT-PCR-ELISA and approximately 106 genomes/reaction for agarose gel detection of the RT-PCR product. The RT-PCR-ELISA assay was used to screen an adult planthopper population collected from an FDV-infected sugarcane field and demonstrated that at least 28% of the insects contained detectable levels of FDV. To address the issue of transovarial transmission, batches of newly emerging planthopper nymphs were assayed using both RT-PCR-ELISA and TaqManTM, and all five batches tested were positive for FDV. The simplest interpretation of these data is that FDV can be transmitted from adult planthoppers to their progeny via the reproductive system. The TaqManTM assay was found to be more sensitive for detection of FDV in biological samples and was able to detect FDV in samples from symptomatic and asymptomatic leaves from infected sugarcane plants; however, RT-PCR-ELISA was not sensitive enough to detect FDV in the asymptomatic leaves. Although quantitative data were obtained by the TaqManTM, with further research it should be feasible to develop routine, quantitative assays for FDV in biological samples.

References
1. Egan BT, Ryan CC, Francki RIB, 1989. Diseases of Sugarcane: Major Diseases, pp. 263-287.
2. Gibson UEM, Heid CA, Williams PM, 1996. Genome Methods 6, 986-994.