Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCRTom Geensa, Angelo Dewittea, Nico Boonb and Daisy Vanrompaya
a Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
b Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
(Received 12 October 2004; accepted 22 March 2005)
Abstract - A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/L DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.
Key words: Chlamydophila psittaci / real-time PCR / species-specific / genotype-specific / diagnosis
Corresponding author: Tom Geens firstname.lastname@example.org
© INRA, EDP Sciences 2005