title: Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes psient1 and psient2 and the selu or seluv gene using PCR-RFLP.
abstract: The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, psient1 and psient2, or the selu or seluv gene. While these two alternative sei-seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or seluv gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3' end of the sei gene through the 5' first quarter of the seln gene allowed pseudogene- and selu- or seluv-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or seluv gene, while selu- or seluv-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or seluv-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei-seln egc locus type.
authors.affiliation: Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Republic of Ireland.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Collery
authors.affiliation: Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Republic of Ireland.
authors.forename: Cyril J
authors.initials: CJ
authors.lastname: Smyth
mesh_terms: D019295:Computational Biology; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021901:DNA, Intergenic / Q000235:genetics; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011544:Pseudogenes; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000145:classification*
chemical_list: D004269:DNA, Bacterial; D021901:DNA, Intergenic; D004768:Enterotoxins; D015247:Deoxyribonuclease HindIII; C102950:endodeoxyribonuclease BccI; D015252:Deoxyribonucleases, Type II Site-Specific; C024412:GGTGA-specific type II deoxyribonucleases