A Simple Random Amplified Polymorphic DNA Genotyping Method for Field Isolates of Dermatophilus congolensis

Abstract

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing ofDermatophilus congolensis

“…In thios study we tested 24 restriction enzymes with 6-8 base-pair recognition sites. Only Sse8387I yielded wellresolved patterns composed of a limited number (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) of fragments, as recommended [72]. Other enzymes tested yielded excessive numbers of small fragments, that were impossible to resolve by PFGE.…”

“…Template DNA was extracted using a fast non-phenolic method as previously described [14]. A total of 21 arbitrary primers were selected from a group of 33 (see Table 2), on the basis of their ability to produce polymorphism, visible as a number of clearly defined electrophoretic bands in trial experiments using a generic RAPD protocol [14]. Sequential optimization of each component of the reaction and the amplification conditions were carried out following a previously described protocol [14].…”

“…Considering its combination of discriminatory power and ease of application, RAPD emerges as a particularly attractive molecular technique especially if the simplified RAPD technique using heated bacterial DNA templates [14] is employed to reduce the time and labour required. This study shows that when an appropriately chosen set of primers is employed, RAPD analysis provides a rapid, reproducible, and powerful genomic typing method for D. congolensis.…”

Characterisation of an extracellular serine protease gene (naspgene) fromDermatophilus congolensis

“…DNA was isolated using a modified version of the protocol described by Wilson [18] with an additional step of 5 h incubation at 37°C with lysozyme (0.2 mg ml −1 ) in Tris‐EDTA buffer, prior to a SDS and proteinase K treatment step, followed by standard phenolic extraction [19].…”

Development of a real-time SYBR Green PCR assay for the rapid detection ofDermatophilus congolensis

“…Genomic DNA was extracted from the cultures using a previously described method [8]. The final DNA pellet was suspended in 25 µL of ultrapure water (Life technologies, Spain) and stored at -20℃ until further use.…”