Limit of Blank Variability as a Determinant of Analytical Sensitivity in a DNA-Binding Dye-Based Digital Polymerase Chain Reaction for the Detection of Chlamydia pneumoniae.

Digital PCR is considered a highly sensitive technique for detecting low-abundance DNA in samples containing excess background DNA. This is particularly relevant for Chlamydia pneumoniae infections, as this obligatory intracellular bacterium can be disseminated by phagocytes from the respiratory tract throughout the whole body. Although persistent bacteria are associated with chronic inflammatory diseases, currently, no diagnostic tools for persistent C. pneumoniae infections exist. In this study, an EvaGreen-based digital PCR assay targeting the chlamydial 16S rRNA gene was initially developed for the QX200 Droplet Digital PCR system; however, poor target recovery, insufficient restriction enzyme digest, and incompatibility with the DNA isolation workflow limited assay performance. Consequently, the assay was redesigned to target the chlamydial groL gene. Analytical performance was determined according to the Clinical and Laboratory Standards Institute (CLSI) EP17-A guideline and the ISO 20395:2019 standard. Notably, the limit of blank varied up to 4-fold between different PCR reagent lots, substantially affecting limit of detection estimates and assay interpretation at low-level. These findings demonstrate the critical importance of assay specification and reagent validation for diagnostics with DNA-binding dye-based assays. Nested PCR exhibited superior analytical sensitivity over the digital PCR assays, yet the limited throughput and increased handling complexity of nested PCR restrict its suitability for routine diagnostic applications. Overall, this study highlights important practical limitations and validation requirements for highly sensitive molecular diagnostic assays.