Multiplex Real-time PCR Assay for the Detection of all Chlamydia Species and Simultaneous Differentiation of C. psittaci and C. pneumoniae in Human Clinical Specimens

We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psit-taci and Chlamydia pneumoniae-two important human respiratory pathogens-in hu-man clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneu-moniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebro-spinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/mu L. The assay results matched with historical assay results for all specimens, ex-cept for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accu-rate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents.