摘要
目的利用环媒恒温扩增技术（loop-mediated isothermal amplification，LAMP），建立检测原型泡沫病毒（prototype foamy virus，PFV）的方法。方法根据PFV的整合酶核心区基因序列设计了3对LAMP引物，利用有链取代活性的Bst DNA聚合酶在63℃对靶序列进行扩增。优化检测条件，建立PFV的检测方法。结果以含目的片段的质粒为模板建立了PFV的检测方法，运用此方法可特异地检测出PFV，而人免疫缺陷病毒、牛免疫缺陷病毒、牛泡沫病毒检测均为阴性，检测灵敏度为50拷贝，较聚合酶链反应高一个数量级。系统可以在15min内完成扩增，同时可以在细胞基因组干扰下检出病毒，对混有病毒基因的人外周血单个核细胞（PBMC）总DNA的检测呈现特异阳性反应。结论建立基于PFV整合酶基因的LAMP检测方法，在PFV感染检测方面有应用前景。
Objective To develop prototype foamy virus（PFV） detection method by loop-mediated isothermal amplification. Methods Three pairs of primers targeting core region of PFV integrase were designed in this study and Bst DNA polymerase was used to amplify target sequence at 63℃. The system was established with all the conditions optimized. Results The method was established with the plasmid containing target sequence as the template. This method could specifically detect PFV infectious clone, no cross- reaction was observed with human immunodeficiency virus infectious clone, bovine immunodeficiency virus infectious clone and bovine foamy virus infectious clone as templates. The detection capability of this system was 50 copy, one order more sensitive than PCR. The amplification could be finished in 15 min and human genomic DNA did not adversely affect the amplification efficiency. Conclusion The PFV detection method by loop-mediated isothermal amplification was established and it had potential usefulness in PFV detection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第2期181-185,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目（30570072,30770097）
关键词
原型泡沫病毒
环媒恒温扩增技术
检测
Prototype foamy virus
Loop-mediated isothermal amplification
Detection
