1887

Abstract

A PCR-based detection system was developed for a pathogen of sugarcane, and other related xanthomonads, using the conserved sequence of two adjacent tRNA genes and the variable length and sequence of the spacer region between them. An appropriate region was identified as follows: tRNA genes with the same anticodon from a wide variety of bacteria were aligned and the most frequent base at each position was chosen to derive primers that would anneal to the gene in either orientation. Pairs of such primers were screened against various species and members of related genera using PCR at low to moderate annealing stringency. A subset of these pairs of tRNA consensus primers gave one or more PCR products which generally displayed interspecific length variability. The primer pair 5'-3' tRNAand 3'-5' tRNAshowed interspecific length polymorphisms between and all other species examined. These PCR products were cloned and sequenced from four isolates of and four isolates from different pathovars of , and the spacer length variation confirmed. Specific tRNA gene primers were derived from the tRNA gene sequences. These primers yielded a PCR product of a characteristic length within most species and pathovars tested. When a primer that projected from tRNAinto the 3' end of the variable intergenic spacer was used with a tRNA-specific primer, PCR was a very sensitive diagnostic test for -infected sugarcane and gave no product or only a faint product with other species of bacteria. The specificity of this PCR-based detection system was further enhanced by a nested PCR reaction that took advantage of the fact that the tRNA-tRNAregion was found to be embedded in a 16S rRNA-23S rRNA gene spacer. By amplifying the region between the 16S rRNA gene and tRNAor between the tRNAand the 23S rRNA gene, the subsequent nested PCR product was shown to be X. -specific.

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1995-12-01
2021-05-13
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