GeXP联合多重PCR系统在腹泻病毒检测中的应用  

GeXP system combined with multiplex PCR technology for detection of diarrhea viruses

作　　者：石莹[1] 陈肖潇[1] 樊学军[1] 田绿波[1] 杨雨[1] 张薇薇[2] 

机构地区：[1]四川出入境检验检疫局国际旅行卫生保健中心,成都610041 [2]成都医学院公共卫生系,610500

出　　处：《中华实验和临床病毒学杂志》2016年第3期319-323,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家质检公益性科研专项（201210046）;国家质检总局科研项目（2014IK064）;四川出入境检验检疫局科研项目（SK201402）

摘　　要：目的 利用GeXP系统结合多重PCR技术，建立诺如病毒（GI和GII组）、轮状病毒、星状病毒的方法。方法根据4种病毒的序列特征，制备病毒标准品质粒，收集病原体样本，利用GenBank下载4种病毒的核苷酸序列，根据4种病毒的序列特征，利用GeXPexpressProfiler设计特异性引物，优化条件使GeXP系统能够特异性、快速的检测对应病毒。评价其灵敏度、特异性及功效，并应用于120份临床标本检测。结果优化后的引物能特异性的检测各病毒，无交叉反应；灵敏度为100％，特异性为99．53％，功效为99．62％，与荧光定量PCR检测结果一致性为99．51％，检测浓度最低至5×10^3／μl拷贝，在已知临床样本灵敏度高、特异性好。结论GeXP方法的建立为快速、准确的检测这4种腹泻病毒提供了便利，对口岸突发群体性腹泻的病原体检出有重要意义。Objective GenomeLab Genetic Analysis System （GeXP） and multiplex-PCR were combined to detect Noroviruse I,II, Astrovirus and Rotavirus. Methods The sequences of these viruses were obtained from Genbank of NCBI, and specific primer pairs were designed by GeXP express Profiler. After optimization, the GeXP system could amplify specific fragments of each virus. The sensitivity and specificity of multiple PCR assay was also evaluated. Optimized assay was further validated with 120 clinical specimens collected from the CDC. Results These primer pairs were successfully used for detecting these viruses at the level of 5 × 10^3copies/ μl without cross reaction. The sensitivity, specificity and efficiency of GeXP assay was 100% , 99.53% and 99.62% , respectively. Comparing to Real-time PCR, the consistence rate reached 99.51% in 120 clinical samples. Conclusions The combined detection of GeXP and multiplex PCR assay could be a high-throughput, rapid and highly specific and sensitive method used in the screening of the diarrhea viruses.

关 键 词：腹泻病毒 GeXP 多重PCR 

分 类 号：R440[医药卫生—诊断学] R512.5[医药卫生—临床医学]