Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP
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 ind...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00643/full |
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| author | Dinka eMandakovic Dinka eMandakovic Benjamín eGlasner Jonathan eMaldonado Jonathan eMaldonado Pamela eAravena Pamela eAravena Mauricio eGonzalez Mauricio eGonzalez Mauricio eGonzalez Verónica eCambiazo Verónica eCambiazo Verónica eCambiazo Rodrigo ePulgar Rodrigo ePulgar Rodrigo ePulgar |
| author_facet | Dinka eMandakovic Dinka eMandakovic Benjamín eGlasner Jonathan eMaldonado Jonathan eMaldonado Pamela eAravena Pamela eAravena Mauricio eGonzalez Mauricio eGonzalez Mauricio eGonzalez Verónica eCambiazo Verónica eCambiazo Verónica eCambiazo Rodrigo ePulgar Rodrigo ePulgar Rodrigo ePulgar |
| author_sort | Dinka eMandakovic |
| collection | DOAJ |
| description | 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. salmonis 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 PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis 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. |
| first_indexed | 2024-12-11T20:44:03Z |
| format | Article |
| id | doaj.art-1b5869b7b5a641659cf6e854a7890430 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-11T20:44:03Z |
| publishDate | 2016-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-1b5869b7b5a641659cf6e854a78904302022-12-22T00:51:24ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-05-01710.3389/fmicb.2016.00643182444Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLPDinka eMandakovic0Dinka eMandakovic1Benjamín eGlasner2Jonathan eMaldonado3Jonathan eMaldonado4Pamela eAravena5Pamela eAravena6Mauricio eGonzalez7Mauricio eGonzalez8Mauricio eGonzalez9Verónica eCambiazo10Verónica eCambiazo11Verónica eCambiazo12Rodrigo ePulgar13Rodrigo ePulgar14Rodrigo ePulgar15Universidad de ChileFondap Center for Genoma Regulation (CGR)Universidad de ChileUniversidad de ChileFondap Center for Genoma Regulation (CGR)Universidad de ChileUniversidad de Chile,Universidad de ChileUniversidad de Chile,Fondap Center for Genoma Regulation (CGR)Universidad de ChileUniversidad de Chile,Fondap Center for Genoma Regulation (CGR)Universidad de ChileUniversidad de Chile,Fondap Center for Genoma Regulation (CGR)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. salmonis 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 PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis 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.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00643/full16S rDNAPCR-RFLPrestriction enzymesPiscirickettsia salmonisComputer-simulation analyses |
| spellingShingle | Dinka eMandakovic Dinka eMandakovic Benjamín eGlasner Jonathan eMaldonado Jonathan eMaldonado Pamela eAravena Pamela eAravena Mauricio eGonzalez Mauricio eGonzalez Mauricio eGonzalez Verónica eCambiazo Verónica eCambiazo Verónica eCambiazo Rodrigo ePulgar Rodrigo ePulgar Rodrigo ePulgar Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP Frontiers in Microbiology 16S rDNA PCR-RFLP restriction enzymes Piscirickettsia salmonis Computer-simulation analyses |
| title | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP |
| title_full | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP |
| title_fullStr | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP |
| title_full_unstemmed | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP |
| title_short | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP |
| title_sort | genomic based restriction enzyme selection for specific detection of piscirickettsia salmonis by 16s rdna pcr rflp |
| topic | 16S rDNA PCR-RFLP restriction enzymes Piscirickettsia salmonis Computer-simulation analyses |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00643/full |
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