【摘要】 目的:建立轮状病毒的荧光定量RT-PCR检测方法,用于检测临床腹泻样本。方法:使用Taqman探针技术,建立轮状病毒VP6特异的荧光定量RT-PCR体系。与WHO推荐的轮状病毒检测方法ELISA平行检测,对该体系的检测范围、灵敏度、特异性等参数进行评价。并将该体系用于临床腹泻样本。结果:该荧光定量RT-PCR体系具有高度灵敏度和特异性,线性范围宽,最低可检出1TCID50/ml。对113份临床腹泻样本的平行检测结果显示,该荧光定量RT-PCR体系与ELISA的检出率差异无统计学意义(χ2=0.41,P>0.5),可同样用于临床样本检测。结论:成功建立了轮状病毒荧光定量RT-PCR体系,该体系灵敏、特异、重复性好,可用于临床检测。更多还原

【Abstract】 Objective: To develop a more sensitive and specific diagnostic test for rotavirus by applying real time quantitative RT-PCR technology.Methods: The real time RT-PCR specific for VP6 gene of rotavirus was developed by applying Taqman technology.And the parameters of the developed real time RT-PCR,including sensitivity,specificity and reproducibility were evaluated.Meanwhile,ELISA was used as a parallel detection method to detect RV in stock and clinical fecal specimen since it was recommended as a standard method for RV detection by WHO.Results: The detection limit of the real time quantitative RT-PCR was 1TCID₅₀/ml,which was 1log more sensitive than ELISA.The relationship between threshold cycle（Ct） and amount of virus was linear（r=0.998） over a range of 1 to 10?000 TCID₅₀/ml.The assay was specific to RV and its efficiency was confirmed by the detection of small amounts of viral RNA in clinical samples.Of the total 113 specimens,80（70%） and 76（67%） were positive by real time RT-PCR and ELISA kit,respectively.The overall detection rate of the assay was not statistically different from that of ELISA（χ² =0.41,P>0.5）.Conclusion: The developed real time RT-PCR assay of group A rotavirus described here allows a large number of samples to be screened rapidly,sensitively and specifically,and it will potentially be a suitable tool for the detection of RV in field,samples including clinical specimen,polluted water,shellfish and so on.更多还原