A multiplex real-time PCR assay for differential identification of avian Chlamydia

Avian chlamydiosis is an acute or chronic disease of birds after infection by Chlamydia. Although Chlamydia psittaci is the primary agent of the disease, two additional species, Chlamydia avium and Chlamydia gallinacea, have also been recognized as potential disease agents. Therefore, the diagnosis of avian chlamydiosis requires differential identification of these avian Chlamydia species. The objective of the present study was to develop a multiplex real-time polymerase chain reaction (PCR) assay to rapidly differentiate between these three species of avian Chlamydia (C. psittaci, C. avium, and C. gallinacea) as well as to detect the genus Chlamydia. Specific genetic regions of the three species were identified by comparative analysis of their genome sequences. Also, the genus-specific region was selected based on 23S rRNA sequences. PCR primers and probes specific to the genus and each species were designed and integrated in the multiplex real-time PCR assay. The assay was highly efficient (94.8-100.7%). It could detect fewer than 10 copies of each target sequence of the genus and each species. Twenty-five Chlamydia control and field DNA samples were differentially identified while 20 other bacterial strains comprising 10 bacterial genera were negative in the assay. This assay allows rapid, sensitive, and specific detection of the genus and the three species of avian Chlamydia in a single protocol that is suitable for routine diagnostic purposes in avian diagnostic laboratories.