Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.
A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.
Heddema
E R
ER
Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. e.r.heddema@amc.uva.nl
Beld
M G H M
MG
de Wever
B
B
Langerak
A A J
AA
Pannekoek
Y
Y
Duim
B
B
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002691:Chlamydophila psittaci / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D010614:Pharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D009956:Psittacosis / Q000175:diagnosis* / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
D017931:DNA Primers