Pseudorabies (PR) is a highly contagious viral disease of great animal health and economic importance in swine industry. The aim of this study was to evaluate different genomic regions, real-time PCR chemistries and equipment for the molecular diagnosis of PR. Eight primer pairs targeting four genes (gB, gC, gE, gD), three different qPCR chemistries (SybrGreen, hydrolysis probes and plexor) and two equipment (ABI7500, Rotorgene 3000) were evaluated. Oligonucleotides targeting gB using hydrolysis probes showed the best performance after evaluating efficiency (99%), the detection limit (10 -1.5 TCID50 mL -1) and diagnostic sensitivity and; therefore, those primers were selected for performance verification factors such as repeatability, reproducibility and robustness (1.39% variance between days, 24% variance between analysts and 4.07% variance in analysis error). The qPCR standardized and validated in this research proved to be reliable for the diagnosis of PR and may be used in diagnostic laboratories that follow ISO 17025 and ISO 16140.
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机译：伪狂犬病（PR）是一种具有高度传染性的病毒性疾病，对养猪业具有重要的动物健康和经济意义。这项研究的目的是评估不同基因组区域，实时PCR化学和设备的PR分子诊断。评估了针对四个基因（gB，gC，gE，gD），三个不同的qPCR化学试剂（SybrGreen，水解探针和plexor）和两个设备（ABI7500，Rotorgene 3000）的八对引物。使用水解探针靶向gB的寡核苷酸在评估效率（99％），检出限（10 -1.5 TCID50 mL -1）和诊断灵敏度后显示出最佳性能;并且因此，选择那些引物作为性能验证因素，例如可重复性，可重复性和鲁棒性（天数差异为1.39％，分析人员差异为24％，分析误差差异为4.07％）。经本研究标准化和验证的qPCR被证明对于PR的诊断是可靠的，可用于遵循ISO 17025和ISO 16140的诊断实验室。
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