title: Identification of Chlamydial species in crocodiles and chickens by PCR-HRM curve analysis.
abstract: Recently, a PCR protocol (16SG), targeting 16S rRNA gene coupled with high resolution melt (HRM) curve analysis was developed in our laboratory and shown to reliably detect and identify the seven different Chlamydiaceae spp. In this study, the potential of this method was assessed for detection and differentiation of Chlamydiosis in clinical specimens. Of the total number of 733 specimens from a range of animal species, 219 (30%) were found positive by 16SG PCR. When a sufficient amount of DNA was available (64 submissions), amplicons generated by the 16SG PCR were subjected to HRM curve analysis and results were compared to that of nucleotide sequencing. In all instances, the infecting Chlamydiaceae spp. was genotyped according to the identity of its nucleotide sequence to a reference species. Analysis of the HRM curves and nucleotide sequences from 16SG PCR amplicons also revealed the occurrence of a Chlamydophila-like, a Parachlamydia-like and a variant of Chlamydophila psittaci in chickens. These results reveal the potential of 16SG PCR-HRM curve analysis for rapid and simultaneous detection and identification of Chlamydiaceae spp. in animals and demonstrate the capacity of this system for rapid identification of new Chlamydiaceae spp. in animals during routine diagnostic testings.
authors.affiliation: School of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria, Australia. tlcooke@unimelb.edu.au
authors.forename: T
authors.initials: T
authors.lastname: Robertson
authors.forename: S
authors.initials: S
authors.lastname: Bibby
authors.forename: D
authors.initials: D
authors.lastname: O'Rourke
authors.forename: T
authors.initials: T
authors.lastname: Belfiore
authors.forename: R
authors.initials: R
authors.lastname: Agnew-Crumpton
authors.forename: A H
authors.initials: AH
authors.lastname: Noormohammadi
mesh_terms: D000489:Alligators and Crocodiles / Q000382:microbiology*; D000818:Animals; D002645:Chickens / Q000382:microbiology*; D002693:Chlamydiaceae / Q000235:genetics / Q000302:isolation & purification*; D002694:Chlamydiaceae Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S