Non-radioactive ribotyping ofHaemophilus ducreyiusing a digoxigenin labelled cDNA probe

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Abstract
Haemophilus ducreyi, the causal organism of chancroid, has increased in significance recently due to its association with HIV transmission. Most previous typing systems have exploited phenotypie characteristics. Detection of ribosomal RNA cistrons, ribotyping, was successfully developed to examine H.ducreyi, but required the use of32P.We have used digoxigenin to define ribotypes from 30 strains ofH. ducreyifrom diverse geographical locations. This was achieved by agarose gel electrophoresis of restriction enzyme (RE) digested DNA extracts. These extracts were vacublotted onto nylon membrane and probed using digoxigenin labelled complementary DNA probe, prepared fromEscherichia coli16S and 23S ribosomal RNA. From 19 REs tested,AvaII,HincII,BglII andBstE II gave clear ribotypes. The ribotypes ofBstE II andBglII used together gave the highest index of discrimination (D = 0·95), 16 types, and showed good reproducibility. This nonradioactive method demonstrates the three important features of a typing system: discrimination, typability and reproducibility.