呼吸系统疾病对猪的健康构成重大威胁,与猪繁殖与呼吸综合征病毒(PRRSV)共感染,猪链球菌(SS),猪圆环病毒2型(PCV2),和猪圆环病毒3型(PCV3)作为主要病原体。因此,PRRSV的精确诊断,PCV2、PCV3和SS在猪呼吸道疾病的预防和控制中至关重要。因此,我们进行了分子生物学信息学分析,以同时检测和区分PRRSV,PCV2、PCV3和SS。我们选择了PRRSV的ORF6基因,PCV2和PCV3的ORF2基因和SS的谷氨酸脱氢酶(GDH)基因为靶标。针对每种病原体设计特异性引物和探针,并对反应条件进行了细致的优化,我们树立了多重TaqMan荧光定量PCR检测办法。随后,我们对这种方法进行了全面评估,评估其特异性,灵敏度,和可重复性。研究结果表明,所建立的多重TaqMan荧光定量PCR检测方法显示出典型的特异性,与其他病原体没有交叉反应的实例。本方法对PRRSV的最低检测浓度,PCV2、PCV3和SS为2.80×101拷贝/微升,1.96×102拷贝/微升,2.30×102拷贝/微升,和1.75×103拷贝/微升,分别。当应用于30个临床样本的分析时,结果与中国标准的PRRSV单向实时qPCR检测方法获得的结果非常相似,以及SS的一般PCR方法,PCV2和PCV3。这项研究强调了强大的特异性,高灵敏度,我们开发的多种TaqMan荧光定量PCR检测方法具有一致的稳定性。它非常适合PRRSV的临床监测,PCV2、PCV3和SS,它在猪群中预防和管理呼吸道疾病的持续努力中具有重要意义。
Respiratory illnesses present a significant threat to porcine health, with co-infections involving Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Streptococcus suis (SS), Porcine Circovirus Type 2 (PCV2), and Porcine Circovirus Type 3 (PCV3) acting as the primary causative agents. As a result, the precise diagnosis of PRRSV, PCV2, PCV3 and SS is of paramount importance in the prevention and control of respiratory diseases in swine. Therefore, we conducted a molecular bioinformatical analysis to concurrently detect and differentiate PRRSV, PCV2, PCV3 and SS. We selected the ORF6 gene of PRRSV, the ORF2 gene of PCV2 and PCV3, and the glutamate dehydrogenase (GDH) gene of SS as targets. Specific primers and probes were designed for each pathogen, and following meticulous optimization of reaction conditions, we established a multiple TaqMan fluorescence quantitative PCR detection method. Subsequently, we subjected this method to a comprehensive assessment, evaluating its specificity, sensitivity, and repeatability. The research results demonstrated that the established multiple TaqMan fluorescence quantitative PCR detection method displays displayed exemplary specificity, with no instances of cross-reactivity with other pathogens. The method\'s minimum detection concentrations for PRRSV, PCV2, PCV3, and SS were 2.80 × 101 copies/µL, 1.96 × 102 copies/µL, 2.30 × 102 copies/µL, and 1.75 × 103 copies/µL, respectively. When applied to the analysis of 30 clinical samples, the results closely mirrored those obtained through Chinese standard uniplex real-time qPCR detection method for PRRSV, as well as the general PCR methods for SS, PCV2, and PCV3. This study underscores the robust specificity, high sensitivity, and consistent stability of the multiple TaqMan fluorescence quantitative PCR detection method that we have developed. It is ideally suited to the clinical monitoring of PRRSV, PCV2, PCV3, and SS, and it carries significant importance in ongoing efforts to prevent and manage respiratory diseases in porcine populations.