Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP
Author
dc.contributor.author
Mandakovic, Dinka
Author
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Glasner, Benjamín
Author
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Maldonado, Jonathan
Author
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Aravena, Pamela
Author
dc.contributor.author
González, Mauricio
Author
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Cambiazo Ayala, Verónica
Author
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Pulgar Tejo, Rodrigo
Admission date
dc.date.accessioned
2016-09-26T15:01:33Z
Available date
dc.date.available
2016-09-26T15:01:33Z
Publication date
dc.date.issued
2016
Cita de ítem
dc.identifier.citation
Frontiers in Microbiology Volumen: 7 Número de artículo: 643 May 2016
es_ES
Identifier
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10.3389/fmicb.2016.00643
Identifier
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https://repositorio.uchile.cl/handle/2250/140507
Abstract
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The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. sairnonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCl for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmon's with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.
es_ES
Patrocinador
dc.description.sponsorship
Fondap Grant Center for Genome Regulation
15090007
Fondecyt
1120254
1151384
3130742