Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

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/µL 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.

Evaluation of a Chlamydophila psittaci Infection Diagnostic Platform for Zoonotic Risk Assessment

“…At the same time, scientist 2 was only positive in nested PCR, but we were unable to type the strain as the CTL/CTU PCR performed directly on clinical specimens remained negative. This is not unusual since the nested PCR is more sensitive than the latter nucleic acid amplification test (3,10). The infection source was traced using the present diagnostic platform and was found to be a parrot relief and breeding center (4).…”

Longitudinal monitoring for respiratory pathogens in broiler chickens reveals co-infection of Chlamydia psittaci and Ornithobacterium rhinotracheale

“…Outer membrane protein A (ompA) genotyping was performed by a C. psittaci genotype-specific real-time PCR (Geens et al, 2005). The latter PCR distinguished genotypes A-F and E/B using genotype-specific primers, genotype-specific probes and competitor oligonucleotides.…”

Zoonotic transmission of Chlamydia psittaci in a chicken and turkey hatchery

“…For all samples, DNA extraction was performed as previously described. Outer-membrane protein A (ompA) genotyping was performed by a C. psittaci genotype-specific real-time PCR (Geens et al, 2005). The test is based on using genotype-specific primers and genotype-specific TaqMan probes.…”