Identification of chlamydial species in crocodiles and chickens by PCR-HRM curve analysis

• Published in: Veterinary microbiology Vol. 145; no. 3; pp. 373 - 379
• Main Authors: Robertson, T., Bibby, S., O’Rourke, D., Belfiore, T., Agnew-Crumpton, R., Noormohammadi, A.H.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26-10-2010; Amsterdam; New York: Elsevier; Elsevier
• Subjects: 16S rRNA gene; Alligators and Crocodiles - microbiology; animal diseases; Animals; bacterial infections; Bacteriology; Biological and medical sciences; chickens; Chickens - microbiology; Chlamydia/ Chlamydophila; Chlamydiaceae; Chlamydiaceae - genetics; Chlamydiaceae - isolation & purification; Chlamydiaceae Infections - diagnosis; Chlamydiaceae Infections - microbiology; Chlamydiaceae Infections - veterinary; Chlamydophila psittaci; crocodiles; diagnostic techniques; disease detection; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Fundamental and applied biological sciences. Psychology; genetic techniques and protocols; high resolution melt curve analysis; HRM curve analysis; methodology; microbial genetics; Microbiology; Miscellaneous; molecular sequence data; new methods; nucleotide sequences; ompA gene; pathogen identification; polymerase chain reaction; Polymerase Chain Reaction - veterinary; rapid methods; ribosomal RNA; RNA, Ribosomal, 16S - chemistry; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; Species identification; Transition Temperature; HRM curve analysis; ompA gene; Chlamydiaceae; Species identification; Chlamydia/ Chlamydophila; 16S rRNA gene; Chlamydia/Chlamydophila; Farming animal; Porin; Poultry; Ribosomal DNA; Identification; Polymerase chain reaction; Vertebrata; Chlamydia; Gene; Chlamydiales; 16S-RNA; Bacteria; Chicken; Aves; Veterinary; Chlamydophila
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2010.04.007

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.