基于颜色判定的环介导等温扩增技术检测HPV6和HPV16  

Colorimetric Detection of HPV6 and HPV16 by Loop Mediated Isothermal Amplification

作　　者：芦春斌[1] 罗乐[1] 杨梦婕[1] 聂凯[2] 王淼[2] 马学军[2] 

机构地区：[1]暨南大学生殖免疫研究所,广州510632 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京102206

出　　处：《病毒学报》2011年第1期64-70,共7页Chinese Journal of Virology

基　　金：广东省科技计划项目(2010B031600108);传染病防治重大专项(2009ZX10004-101;2008ZX10004-002;001;004)

摘　　要：建立了一种基于颜色判定的简单、快速和灵敏的检测方法,即环介导等温核酸扩增技术(LAMP)应用于HPV6和HPV16亚型的检测。该技术设计分别对应于HPV6和16的E6和E7基因序列中6个区段的4条特异引物,在等温条件下(63℃)进行核酸扩增反应1h,在扩增前加入HNB染料(羟基萘酚蓝)作为反应指示剂,以HNB染料颜色的变化做为结果判定的标准,并经LA-320实时浊度仪和琼脂糖电泳证实。文中利用这种技术对13份已知的单重感染13种HPV不同亚型的临床样本进行了特异性分析,同时对2个含目的片段的克隆质粒的系列稀释物进行了灵敏度分析。结果显示LAMP方法特异性高,颜色法判断的灵敏度均为1000个拷贝DNA分子水平,比Real-time PCR低10～20倍,对62份宫颈刮片临床标本的HPV6和HPV16检出率和HPV分型试剂盒一致。因此,基于颜色判定的环介导等温扩增方法有望应用于人乳头瘤病毒HPV6和HPV16感染的快速筛选,具有在基层疾病预防控制中心和医院推广和应用的潜力。A simple, rapid and sensitive colorimetric Loop Mediated Isothermal Amplification （LAMP） method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63℃ for one hour. The amplification process of LAMP was monitored by the addition of HNB （hydroxy naphthol blue） dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by Real-time turbidimeter and agarose eleetrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1,000 copies, 10-20 times lower than that of Real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.

关 键 词：等温扩增 人乳头瘤病毒(HPV) 

分 类 号：R373.1[医药卫生—病原生物学] R446[医药卫生—基础医学]