中国猪业 ›› 2025, Vol. 20 ›› Issue (5): 65-72.doi: 10.16174/j.issn.1673-4645.2025.05.0008

• 生物安全与疫病防控 • 上一篇    

PDCoV N基因TaqMan荧光定量PCR检测方法的建立

马翮斯,罗灵芝,王元红,虞凌雪,李国新   

  1. 中国农业科学院上海兽医研究所,上海200241
  • 出版日期:2025-10-25 发布日期:2025-10-25

  • Online:2025-10-25 Published:2025-10-25

摘要: 猪德尔塔冠状病毒(Porcine Deltacoronavirus, PDCo V)是一种对哺乳仔猪致病性较强的病原体,感染后可导致急性水样腹泻、呕吐以及高死亡率(30%~50%),对全球养猪业危害严重。为建立一种灵敏且准确的PDCoV检测方法,本研究基于PDCoV N基因的保守序列设计特异性引物和探针,优化反应体系,成功构建了一种TaqMan荧光定量PCR(qPCR)检测方法。以PDCoVN基因重组质粒作为标准品,该方法在8.79×101~8.79×108copies/μL范围内线性关系良好(R2=0.997,斜率=-3.236),最佳引物和探针浓度分别为0.4μmol/L和0.2μmol/L。结果显示,该方法敏感性高,最低检测限为8.79 copies/μL;特异性强,与猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)、伪狂犬病病毒(Pseudorabies virus, PRV)、猪繁殖与呼吸障碍综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)均无交叉反应;重复性好,组间变异系数和组内变异系数均小于1.5%。本研究建立的qPCR方法可为PDCoV的临床检测和流行病学调查提供可靠的技术支撑。

关键词: 猪;猪德尔塔冠状病毒;核衣壳基因;TaqMan qPCR;仔猪腹泻

Abstract: Porcine deltacoronavirus (PDCoV) is a highly pathogenic virus that causes acute watery diarrhea, vomiting and high mortality (30%~50%) in suckling piglets, posing a significant threat to the global swine industry. To develop a sensitive and accurate detection method for PDCoV, this study designed specific primers and probes targeting the conserved region of the PDCoV nucleocapsid (N) gene, optimized the reaction system, and successfully established a TaqMan-based fluorescence quantitative PCR (qPCR) assay. Using a recombinant plasmid containing the PDCoV N gene as a standard, the method exhibited a strong linear correlation within the range of 8.79×101 to 8.79×108 copies/μL (R2=0.997, slope=-3.236), with optimal primer and probe concentrations of 0.4 μmol/L and 0.2 μmol/L, respectively. The assay demonstrated high sensitivity, with a detection limit of 8.79 copies/μL; excellent specificity, with no cross-reactivity observed with Porcine Epidemic Diarrhea Virus (PEDV), Pseudorabies Virus (PRV), or Porcine Reproductive and Respiratory Syndrome Virus (PRRSV); and good reproducibility, with inter- and intra-assay coefficients of variation below 1.5%. The qPCR method developed in this study provides a reliable technical platform for the clinical diagnosis and epidemiological surveillance of PDCoV.

Key words: pig; PDCoV; N gene; TaqMan qPCR; piglet diarrhea

中图分类号:  S828;S852.65+1

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