Non-radioactive ribotyping of Haemophilus ducreyi using a digoxigenin labelled cDNA probe.
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 phenotypic characteristics. Detection of ribosomal RNA cistrons, ribotyping, was successfully developed to examine H. ducreyi, but required the use of 32P. We have used digoxigenin to define ribotypes from 30 strains of H. ducreyi from 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 from Escherichia coli 16S and 23S ribosomal RNA. From 19 REs tested, Ava II, Hinc II, Bgl II and BstE II gave clear ribotypes. The ribotypes of BstE II and Bgl II used together gave the highest index of discrimination (D = 0.95), 16 types, and showed good reproducibility. This non-radioactive method demonstrates the three important features of a typing system; discrimination, typability and reproducibility.
Department of Medical Microbiology, St Mary's Hospital Medical School, Paddington, London.
T J
TJ
Brown
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CA
Ison
D015373:Bacterial Typing Techniques; D015342:DNA Probes; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004076:Digoxigenin; D004587:Electrophoresis, Agar Gel; D006191:Haemophilus ducreyi / Q000145:classification* / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012335:RNA, Ribosomal / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping
D015342:DNA Probes; D004269:DNA, Bacterial; D012335:RNA, Ribosomal; D004262:DNA Restriction Enzymes; D004076:Digoxigenin