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PubMedDate not supplied

Detection of PCR products from Mycobacterium avium subspecies Paratuberculosis using oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA.

Bacterial Typing Techniques↗

PubMedDate not supplied

Molecular typing of avian Escherichia coli isolates by random amplification of polymorphic DNA.

Escherichia coli is a common inhabitant of the gastrointestinal tract of most animals. Like most pathogenic E. coli, avian isolates cannot be distinguished biochemically from the normal commensals inhabiting the gastrointestinal tract of birds. Using a molecular approach, we were able to identify genetic differences among avian E. coli isolates by restriction fragment length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). Several different RFLPs were observed among avian E. coli isolates using DNA probes for 16S ribosomal RNA genes (rrn) and insertion sequence elements (IS2). We were also able to observe differences in DNA banding patterns generated by RAPD analysis. Similarities and differences among avian E. coli were discernible using RFLPs and RAPD analysis, whereas conventional bacteriological methods failed to differentiate these isolates. Based on RAPD patterns, avian E. coli appear to be genetically diverse. Of 16 different RAPD types (RT) encountered, 84% of E. coli fell into seven major RTs. One RT was present in clinical isolates but absent from the commensals isolated in this study. Many of these different E. coli RTs were not geographically restricted to northern Georgia but were also observed in other southern states in the United States. Resistance to various antibiotics was randomly associated with different E. coli RTs. Sarafloxacin resistance was present among different E. coli RTs, suggesting that antibiotic usage is not selecting for a clonal population in avian E. coli. RAPD provides a rapid and powerful tool to study the epidemiology of avian E. coli.

Animals↗