牛冠状病毒纳米PCR检测方法的建立及初步应用

Establishment and preliminary application of a nano-PCR method for detection of bovine coronavirus

为了更加快速地检测牛冠状病毒(BCoV),本试验根据BCoV的具有较高保守性的N基因序列设计1对特异性引物,结合纳米金颗粒材料,通过反应条件和体系优化,建立一种新型的快速灵敏的BCoV检测方法,并应用于临床检测。特异性试验与敏感性试验结果表明,本试验建立的nano-PCR方法对BCoV特异,与牛轮状病毒(BRV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛呼吸道合胞体病毒(BRSV)、牛副流感病毒3型(BPIV3)均无交叉反应。nano-PCR的敏感性是普通PCR的100倍,普通PCR最低核酸检测量为2.24×10^(3)copies/μL,nano-PCR最低核酸检测量为2.24×10^(1)copies/μL。应用所建立的方法对采集的43份腹泻牛的粪拭子、鼻拭子、肺部组织样品进行检测,检出BCoV阳性样品17份,普通PCR检出阳性样品11份,两种方法的符合率为100%。此次建立的nano-PCR在检测牛冠状病毒时具有更高的特异性和敏感性,对临床上的快速诊断及检测具有一定的意义。

In order to detect bovine coronavirus(BCoV) and confirm the diagnosis of bovine coronavirus disease more rapidly,based on the high conserved N gene sequence of BCo V,a pair of specific primers were designed,a new rapid and sensitive detection method was established for detecting BCo V by combinding the nanogold particle materials and the optimization of reaction conditions and system,and was applied to clinical detection.The results of the specificity and sensitivity tests showed that the nano-PCR assay established in this experiment was specific for BCo V and had no cross-reactivity with BRV,BVDV,IBRV,BRSV and BPIV3.The detection limit of BCo V nano-PCR(2.24×10^(1) copies/μ L) was 100-fold more sensitive than that of the common PCR(2.24×10^(3) copies/μ L).The established method was applied to43 fecal,nasal swab and lung tissue samples collected from cattles with diarrhea,17 BCoV positive samples were detected by the nano-PCR,and 11 positive samples were detected by common PCR.The compliance rate of the two methods was 100%.This established nano-PCR has higher specificity and sensitivity in the detection of bovine coronavirus,which is of significance for rapid diagnosis and detection in clinical settings.