Restriction Fragment Length Polymorphism Analysis of Citrus Tristeza Virus Isolates in Japan and its Application to Cross-protection Experiments

摘要

Several primer pairs were designed between open reading frame 6 and the 3' untranslated region for amplification of Citrus tristeza vims (CTV) complementary DNA (cDNA). Reverse transcription polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the PCR products using an appropriate combination (if primer pairs and restriction enzymes was shown to be useful for distinguishing diverse CTV isolates in Japan. Three distinct experiments to investigate cross-protection against severe CTV infection were carried out using two mild isolates (M15A and M23A), The mild isolate M15A was previously reported to be a promising protective isolate. M15A, however, cannot be distinguished from severe isolates by means of existing monoclonal antibody techniques. Our work also demonstrates that RT-PCR-RFLP can be successfully applied to evaluation of the protective ability of mild isolates M15A and M23A against severe CTV isolates, since the results of RT-PCR-RFLP coincided with those of symptom observations in the cross-protection experiments.