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

We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all species and simultaneous differentiation of and -two important human respiratory pathogens-in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all species, , and . To validate the assay, we used a panel of 49 culture isolates comprising seven genotypes, eight isolates, seven other 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 cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for or . The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/μL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of infections and provide a greater understanding of the burden of disease caused by these agents.