鸭星状病毒3型TaqMan探针荧光定量PCR检测方法的建立与应用

Development and application of a TaqMan real-time PCR assay for duck astrovirus-3

根据已报道的鸭星状病毒3型(duck astrovirus-3,DAstV-3)毒株的nsp1a基因序列设计并合成特异性引物和探针,经优化反应条件建立一种以nsp1a基因为靶标的检测DAstV-3的TaqMan实时荧光定量PCR方法。结果显示,该方法的Ct与标准品模板浓度在2.13×10^(1)~2.13×10^(7)拷贝/μL之间线性关系良好,相关系数R~2和扩增效率分别为0.997和1.09;该方法仅特异性检出DAstV-3核酸,而对其他鸭常见病毒核酸无扩增反应;最低检测下限为21.30拷贝/μL;组内和组间重复试验变异系数分别为0.58%~2.19%和0.68%~2.53%,均低于5.00%;利用所建方法对临床采集的231份疑似DAstV-3感染病料进行检测,发现DAstV-3的阳性率为57.10%(132/231)。结果表明,本试验所建方法操作简便、特异、敏感、重复性好,为DAstV-3的临床检测和流行病学调查提供技术支持。

To establish the TaqMan real-time PCR assay for detection of duck astrovirus-3(DAstV-3),an emerging infectious disease pathogen inducks,one pair of specific primers and probes were designed based on the nsp1a gene of DAstV-3 deposited in GenBank.The TaqMan real-time PCR assay for detecting DAstV-3 was successfully developed by optimizing the reaction conditions.The standard curve showed a good linear relationship between Ct and the template concentrations in the range of 2.13×10^(1)-2.13×10^(7) copies/μL,and the value of the correlation coefficient R~2 and amplification efficiency were 0.997 and 1.09,respectively.Besides,the assay had good specificity for DAstV-3,and had no cross-reaction with other common infectious disease pathogens in ducks.The minimum detection limit of the assay was 21.30 copies/μL for DAstV-3.The coefficient of variations(CV)in intra-and inter-assays were 0.58%-2.19%and 0.68%-2.53%,both less than 5.00%,indicating that the method had a good repeatability.A total of 231 suspected DAstV-3-positive clinical samples were detected by the established assay,and the DAstV-3 positive rate was 57.10%(132/231).The established TaqMan real-time PCR assay is simple,specific,sensitive and reproducible,which provides simple and effective technical support for clinical detection and epidemiological investigation of DAstV-3.