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Updated: Sep 8, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Published on: August 6, 2011
Application of a nested, multiplex PCR to psittacosis outbreaks
T O Messmer1, S K Skelton, J F Moroney
1National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, Department of Health and Human Services, Atlanta, Georgia 30333, USA. tym2@ciddbd2.em.cdc.gov
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.
Insights
A new nested, multiplex PCR method enables simultaneous detection of three key chlamydiae species. This assay enhances sensitivity for diagnosing chlamydial infections in both human and avian samples.
Area of Science:
- Microbiology
- Molecular Biology
- Veterinary Medicine
Background:
- Chlamydiae are significant bacterial pathogens affecting both humans and animals.
- Accurate and sensitive detection methods are crucial for diagnosing and controlling chlamydial infections.
- Existing diagnostic methods may face limitations with sensitivity and inhibitors in clinical specimens.
Purpose of the Study:
- To develop a highly sensitive, nested, multiplex PCR assay for the simultaneous detection of three Chlamydia species.
- To differentiate between *Chlamydia pneumoniae*, *Chlamydia psittaci*, and *Chlamydia trachomatis*.
- To improve the detection rate of *Chlamydia psittaci* in avian specimens.
Main Methods:
- Development of a two-step nested, multiplex PCR targeting the 16S rRNA gene.
- The first PCR step was genus-specific for Chlamydiae.
- The second PCR step was multiplexed to differentiate between *C. pneumoniae*, *C. psittaci*, and *C. trachomatis* based on amplicon size.
Main Results:
- The assay demonstrated a limit of detection of 5 inclusion-forming units for each PCR step.
- The method successfully identified *C. psittaci* transmission from pet store birds to humans during an outbreak.
- PCR application to avian specimens significantly increased the detection rate of *C. psittaci* compared to traditional culture methods.
Conclusions:
- The developed nested, multiplex PCR is a sensitive and specific tool for simultaneous detection of three chlamydiae species.
- This assay is effective in identifying *C. psittaci* in both human and avian samples, aiding in outbreak investigations.
- The method offers improved diagnostic capabilities for chlamydial infections, particularly in avian populations.
