College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Braz J Microbiol. 2022 Mar;53(1):359-368. doi: 10.1007/s42770-021-00633-w. Epub 2021 Oct 28.
It is laborious to diagnose the infections of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Suid herpesvirus 1 (SuHV-1) because of the similar clinical symptoms in piglets. Staphylococcus aureus (S. aureus), Streptococcus suis (S. suis), Salmonella choleraesuis (S. choleraesuis, serotype: 6,7:c:1,5), and Escherichia coli (E. coli) are common secondary bacterial pathogens in viral infections. Furthermore, the mixed infection of these viral and bacterial pathogens is more and more common in practical swine breeding. Therefore, a TaqMan multiplex qPCR method for simultaneous detection and differentiation of their pathogen was established in this study by designing specific primers and probes for the E2 gene of CSFV, the ORF7 gene of PRRSV, the ORF1 gene of PCV2 and the gE gene of SuHV-1, the nuc gene of S. aureus, the ef-tu gene of S. suis, the ivnA gene of S. choleraesuis, and the 23S rRNA gene of E. coli, and its specificity, sensitivity, and reproducibility were subsequently tested. The results showed that TaqMan multiplex qPCR method showed a high specificity with no cross reaction between different viruses, and a good repeatability with its coefficient of variation lower than 5%. Besides, the sensitivity of this method was also at least 10 times higher compared with conventional PCR. Overall, this study provided a reliable multiplex TaqMan qPCR method for the diagnosis and differentiation of the mentioned pathogens in pigs, laying a certain technical basis for disease prevention and control.
诊断猪瘟病毒（Classical swine fever virus，CSFV）、猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）、猪圆环病毒 2 型（Porcine circovirus type 2，PCV2）和猪疱疹病毒 1 型（Suid herpesvirus 1，SuHV-1）感染较为困难，因为仔猪的临床症状相似。金黄色葡萄球菌（Staphylococcus aureus，S. aureus）、猪链球菌（Streptococcus suis，S. suis）、猪霍乱沙门氏菌（Salmonella choleraesuis，S. choleraesuis，血清型：6，7：c：1，5）和大肠杆菌（Escherichia coli，E. coli）是病毒感染中常见的继发性细菌病原体。此外，这些病毒和细菌病原体的混合感染在实际养猪中越来越常见。因此，本研究通过设计 CSFV 的 E2 基因、PRRSV 的 ORF7 基因、PCV2 的 ORF1 基因和 SuHV-1 的 gE 基因、S. aureus 的 nuc 基因、S. suis 的 ef-tu 基因、S. choleraesuis 的 ivnA 基因和 E. coli 的 23S rRNA 基因的特异性引物和探针，建立了一种用于同时检测和区分这些病原体的 TaqMan 多重 qPCR 方法，并对其特异性、敏感性和重复性进行了测试。结果表明，TaqMan 多重 qPCR 方法具有较高的特异性，与不同病毒无交叉反应，重复性好，变异系数低于 5%。此外，该方法的灵敏度比常规 PCR 至少高 10 倍。总的来说，本研究为猪病的诊断和鉴别提供了一种可靠的多重 TaqMan qPCR 方法，为疾病的防控奠定了一定的技术基础。
