Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans |
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Authors: | Tony HS Woo BKC Patel Lee D Smythe Meegan L Symonds Michelle A Norris Michael F Dohnt |
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Institution: | School of Biomolecular and Biomedical Sciences, Faculty of Science and Technology, Griffith University, Nathan Campus, Brisbane, Qld. 4111, Australia;WHO/FAO Collaborating Centre for Reference and Research on Leptospirosis, P.O. Box 495, Brisbane, Qld. 4000, Australia |
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Abstract: | Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular Dde I restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans . Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the Dde I restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans . We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa , weilii , and inadai , but also simplified a previous PCR-RFLP protocol. |
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Keywords: | Leptospira Allele-specific amplification Polymerase chain reaction-restriction fragment length polymorphism 16S rRNA gene |
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