Development and Validation of a New TaqMan Real-Time PCR for the Detection of .

(ORT) has been associated with poultry respiratory disease worldwide. The organism is fastidious and isolation is challenging. One TaqMan real-time PCR (qPCR) assay has been developed for the detection of ORT. However, during validating the ORT qPCR, the assay performance was suboptimal. During the in silico evaluation, deviations from the basic parameters for primers and probes designs (e.g., presence of stable undesirable primer-dimers) were observed. The suboptimal design led to low efficiency and low sensitivity of the assay. Initially, modification on the probe was carried out to improve the performance of the assay. However, the assay's performance (efficiency and sensitivity) was still suboptimal. In this manuscript, we describe the development of a new qPCR assay and the comparison of its performance with the currently available assay. A highly efficient, sensitive, and specific qPCR assay was developed with approximately 1000-folds reduction in the limit of detection (from 3 × 10 plasmid DNA copies/mL to 1 × 10 plasmid DNA copies/mL). Additionally, the efficiency of the new assay (E = 98.70%) was significantly better than the current assay (E = 73.18%). The newly developed assay is an improved diagnostic tool for the sensitive and efficient diagnosis of ORT from clinical samples.