摘要
目的建立灵敏、特异的诺如病毒基因Ⅱ型的荧光定量RT-PCR方法,用于临床腹泻粪便样本病毒的定量检测。方法构建质粒DNA,并转录合成RNA作为标准品,建立和优化诺如病毒基因Ⅱ型荧光定量RT-PCR方法和反应体系,评价该方法的灵敏度、特异性、重复性,并进行临床粪便样本的检测。结果此方法最低可以检出102拷贝数/μl,能特异地检出诺如病毒基因Ⅱ型,与诺如病毒基因Ⅰ型无交叉反应,与柯萨奇病毒B组、脊髓灰质炎病毒、肠道病毒、星状病毒、甲肝病毒、埃可病毒和轮状病毒无交叉反应。针对标准品,2次批内试验的荧光信号循阈值(Ct值)变异系数(CV)分别为1.60%、0.70%,2次批间试验的变异系数(CV)分别为0.40%、0.40%,均在5%以下。结论本研究建立的实时定量RT-PCR检测诺如病毒基因Ⅱ型的方法,其灵敏度、特异性和重复性良好,可用于该病毒的快速检测。
Objective To establish a sensitive and specific real-time reverse transcription-PCR for rapid detection and quantitation of Norovirus genogroup Ⅱ （ NoV G Ⅱ ） in diarrhea stool samples,water,or food. Meth- ods The construction of a plasmid DNA, transcription and synthesis of the RNA were carried out;the assay for NoV G Ⅱ was established and optimized based on real-time fluroescent quantitative RT-PCR. The sensitivity, specificity and reproducibility of the reaction system were evaluated;and some clinical stool samples were detected by this method. Results The minimum detection limit of the method was 102 copies/pal. This method could specifically de- tect the NoV G Ⅱ that no cross-reaction to Norovirus G I , Coxsackie virus group B, Polio virus, Enterovirus, Astro- virus, Hepatitis A virus, Echo virus, and Rotavirus was observed. The coefficient of variation （CV） of cycles thresh- old（Ct） value of fluorescent signal was 1.60% ,0.70% in two times intra-batch assay ; and 0.40% , 0.40% in inter-batch assay ; all of them were below 5 %. Conclusion The method which we have set up has good sensi- tivity, specificity, and reproducibility. It could be applied to rapid detection of Norovirus genogroup Ⅱ in public health emergencies.
出处
《解放军预防医学杂志》
CAS
2013年第3期200-203,共4页
Journal of Preventive Medicine of Chinese People's Liberation Army
基金
国家自然基金重点项目(No.30930078)
军队"十二五"面上项目(No.CWS11C090)
