基于TaqMan探针的A群轮状病毒荧光RT-PCR检测方法的建立及应用

Establishment and application of a TaqMan probe-based fluorescent RT-PCR method for the detection of group A rotaviruses

摘要:

背景 A群轮状病毒(group A rotavirus, RVA)是导致婴幼儿及幼龄动物发生腹泻的主要人兽共患病病原之一，具有较强的传染性。目的 建立一种快速、特异检测猪、牛、羊、人等物种RVA的实时荧光定量RT-PCR检测方法。方法 基于GenBank中的猪、牛、羊和人源RVA NSP5基因保守序列，设计1对特异性引物、1条探针，该探针带有TaqMan-MGB发光基团，优化最适退火温度(T_m)、最适引物和探针浓度，建立一种可以检测猪、牛、羊和人等物种RVA的实时荧光定量RT-PCR检测方法。结果 建立的RVA荧光定量RT-PCR方法最佳退火温度为51 ℃；最适引物和探针浓度均为0.4 μmol/L；敏感性高，最低检测限可达3.24 copies/μL；特异性强，与猪、牛、羊等常见病原无检测交叉反应；重复性好，其批内和批间试验变异系数分别为0.063%-0.812%和0.612%-1.016%，可以快速、特异检测猪、牛、羊样品和人轮状病毒，有很好的临床适用性。结论 本实验建立了一种检测多物种RVA的实时荧光定量RT-PCR方法，为猪、牛、羊和人等多物种的RVA流行病学调查和早期诊断提供了良好的技术支撑。

Abstract:

Background Group A rotavirus (RVA) is one of the primary zoonotic pathogens responsible for diarrhea in infants, young children, and young animals, exhibiting strong infectivity.Objective To establish a rapid, specific method for the detection of RVAs of porcine, bovine, ovine, and human origins.Methods A pair of specific primers and one probe with TaqMan-MGB were designed based on the conserved sequence of the NSP5 gene of porcine, bovine, ovine, and human RVAs published in GenBank. A real-time fluorescent quantitative RT-PCR method capable of detecting porcine, bovine, ovine, and human RVAs was established after optimization of the annealing temperature (T_m) and primer and probe concentrations.Results The optimal T_m, primer concentration, and probe concentration of the established real-time fluorescent quantitative RT-PCR method were 51 ℃, 0.4 μmol/L, and 0.4 μmol/L, respectively. The method had high sensitivity, with the lowest limit of detection reaching up to 3.24 copies/μL. It had strong specificity, without cross-reactivity with other common porcine, bovine, and ovine pathogens. The method demonstrated good reproducibility, with the intra- and inter-group coefficients of variation being 0.063%-0.812% and 0.612%-1.016%, respectively. Moreover, the method showed excellent clinical applicability for the detection of porcine, bovine, ovine, and human samples.Conclusion A real-time fluorescent quantitative RT-PCR method was established in this study, providing technical support for the epidemiological investigation and early diagnosis of RVAs of porcine, bovine, ovine, and human origins.