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Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.