The generation of specific DNA primers using random amplified polymorphic DNA and its application to Verticillium dahliae

A DNA fragment apparently unique to Verticillium dahliae was found by compa ring RAPD profiles of V. dahliae to those of other closely related fungi. R APD analyses were performed on eight V. dahliae, six V. albo-atrum and thre e V. tricorpus isolates to identify DNA sequences specific to V. dahliae. R APD primer E20 (AACGGTGACC) yielded a 567 bp band shared only by V. dahliae isolates. This band from isolate V14 was cloned and sequenced. No signific ant sequence similarity was found between this amplicon and any other nucle ic acid sequence in the databases. Based on the sequence information, a pai r of PCR primers, VDS1 (5'-CACATTCAGTTCAGGAGACGGA-3') and VDS2 (5'-CCTTCTAC TGGAGTATTTCGG-3') was designed. PCR tests showed that VDS1 and VDS2 amplifi ed the expected fragment of DNA from 62 V. dahliae isolates from diverse ho sts and geographical origins, but not from any other sources of DNA tested, including DNA from the most closely related species, V. albo-atrum. Southe rn blot analysis showed that the PCR product specifically hybridized to V, dahliae genomic DNA. An internal control was constructed for competitive PC R and used to develop a detection and quantification assay for V. dahliae, for which the detection limit was determined.