荧光定量RT-PCR在轮状病毒检测中的应用

Quantitation of rotavirus by real time reverse transcription polymerase chain reaction

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。

Objective： To develop a more sensitive and specific diagnostic test for rotavims 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 llog 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 （ χ^2 = 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.