Abstract
Ornithobacterium rhinotracheale (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 × 106 plasmid DNA copies/mL to 1 × 103 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.
Highlights
Bacterial respiratory infections are one of the most significant challenges facing the poultry industry in the USA and worldwide [1,2]
The primers and probe of the newly developed assay were designed to target a specific segment within the 16S ribosomal ribonucleic acid (rRNA) gene
As a first step in the in silico analysis of all primers and probes included in this study, BLAST specificity analysis was conducted
Summary
Bacterial respiratory infections are one of the most significant challenges facing the poultry industry in the USA and worldwide [1,2]. They lead to significant economic losses due to high condemnation rates, increased mortality, and production losses [2,3]. Ornithobacterium rhinotracheale (ORT) is one of the most important bacterial respiratory pathogens primarily facing the turkey industry. O. rhinotracheale affects turkeys, chickens, and other domestic and wild avian species [7]. The clinical picture of ornithobacteriosis in turkeys and chickens is not pathognomonic, and ORT presents itself similar to many other respiratory diseases caused by bacterial or viral pathogens [5,7]
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