|
|
|
|
|
|
Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding. M. C. Pearce, 2004.The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E . coli O157 in immunomagnetic separation (IMS)-positive fecal samples . The density of E . coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g–1 between samples from the same fecal pat . The density in most positive samples was <100 CFU g–1, the limit of reliable detection by IMS . Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50% . It is therefore probable that most surveys have greatly underestimated the prevalence of E . coli O157 shedding in cattle and the proportion of farms with shedding cattle . The sensitivity of the detection of E . coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample . Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis. Manuela Giovannetti, 2003.We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae . Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85% . Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges . No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts . Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae . Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage . The intraspecific genetic diversity of G . mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates . Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts . Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry. Dario De Medici, 2003.The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples . First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared . The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system . The specificity of the reaction was determined by the melting temperature (Tm) of the amplicon obtained . The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001) . The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles) . Boiling was selected as the preferred extraction method because it is the simplest and most rapid . This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2 . The specificity of the reaction was confirmed by the Tm, which was consistently specific for the amplicon obtained; the mean peak Tm obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C . The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 103 to 108 CFU/ml) showed good linearity (R2 = 0.9767) and a sensitivity limit of less than 103 CFU/ml . The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
