Development and Validation of TaqMan Real-Time Quantitative PCR Assay for Improved Diagnosis of Spotty Liver Disease

Spotty liver disease (SLD) is a reemerging infection caused by Campylobacter species that results in increased mortality and reduced egg production, with increased incidence in cage-free commercial layer hens. Recently, Campylobacter hepaticus has been identified as a key pathogen responsible for SLD. The laboratory diagnosis of SLD primarily relies on isolating C. hepaticus colonies, a process hindered by the bacterium's fastidious nature and the requirement for time-consuming and specialized culture conditions. Molecular diagnosis using quantitative real-time PCR (qPCR) overcomes these limitations and offers a more sensitive and specific alternative. However, the existing qPCR assay using a DNA-binding dye chemistry suffers from nonspecific binding to any double-stranded DNA in the sample, which could potentially lead to an increased incidence of false-positive cases. In this study, we present the development of a more specific TaqMan probe-based qPCR assay targeting the glycerol kinase gene of C. hepaticus. This assay demonstrated excellent analytical specificity and sensitivity, with a detection limit of 5 copies/µl and a PCR efficiency of 95.15%. Additionally, it exhibited 100% diagnostic specificity and sensitivity. Furthermore, probe-based PCRs are the most commonly used type of diagnostic PCR assay and are much better suited for routine diagnostics compared to other types of PCRs. In conclusion, the newly developed assay represents a significant advancement in the accurate and efficient diagnosis of SLD caused by C. hepaticus directly from clinical samples.