Application of a nested, multiplex PCR to psittacosis outbreaks

Abstract

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

Characterization ofChlamydiaceaespecies using PCR and high resolution melt curve analysis of the 16S rRNA gene

“…Oligonucleotide sets CTU/CTL (Denamur et al. 1991) and 16S rRNA (Messmer et al. 1997) failed to generate a visible band on agarose gel when tested against a number of Chlamydiaceae reference species as shown in Table 3.…”

“…The PCRs using the oligonucleotide sets CTU/CTL (Denamur et al. 1991), 16S rRNA (Messmer et al. 1997), 16SIG, 16SF2/23SIG (Everett et al.…”

“…1999) and 16SG PCR were performed in 25 μl total reactions containing 2 μl of extracted DNA, 1 μl of each of 1·25 mmol l −1 dATP, dTTP, dGTP and dCTP (Promega), 2 μl of 25 mmol l −1 MgCl 2, 2 μl of each 25 μmol l −1 forward and reverse oligonucleotides, 0·3 μl of Taq DNA polymerase (Promega), 5 μl 5× Go Taq flexi green buffer (Promega) and 2·5 μl of 5 μmol l −1 SYTO 9 green fluorescent nucleic acid stain (Invitrogen). The 16S rRNA (Messmer et al. 1997) and 16SG PCR reactions were subjected to an initial denaturing period of 2 min at 95°C, and then to 35 cycles of 20 s at 96°C, 20 s at 58°C (annealing temperature) and 20 s at 72°C, followed by a final extension period of 2 min at 72°C.…”

“…2004; Condon and Oakey 2007) have demonstrated that a family or genus‐specific oligonucleotide set targeting the 16S rRNA gene can detect a wide range of Chlamydiaceae species; however, the individual species cannot be distinguished from one another solely by subjecting the amplicons to electrophoresis through an agarose gel. A number of studies (Messmer et al. 1997; Everett and Andersen 1999; Devereaux et al.…”

“…2006; Condon and Oakey 2007) have investigated other techniques including nested PCR, quantitative real‐time PCR and RFLP to differentiate Chlamydiaceae to species level. While nested PCR using a family‐specific amplification followed by a species‐specific amplification or probe has been shown to clearly distinguish Chlamydiaceae species, the technique is time‐consuming and requires separate reactions for each species tested (Messmer et al. 1997).…”