免疫磁珠分离与实时定量PCR技术联合检测水中轮状病毒的研究  

Rapid Detection of Rotavirus in Water Samples Using Immunomagnetic Separation Combined with Real Time PCR

作　　者：杨万[1] 何苗[1] 李丹[1] 施汉昌[1] 刘丽[1] 

机构地区：[1]清华大学环境科学与工程系,环境模拟与污染控制国家重点联合实验室,北京100084

出　　处：《环境科学》2009年第5期1368-1375,共8页Environmental Science

基　　金：国家重点实验室专项基金项目(08Y01ESPCT)

摘　　要：建立了一种免疫磁珠分离技术联合实时定量PCR快速定量检测水中轮状病毒的方法.通过制备能够分离水中轮状病毒的特异免疫磁珠,优化分离条件,建立了免疫磁珠分离前处理方法,并与逆转录、实时定量PCR结合,成功用于水中轮状病毒的检测.研究发现,在1 mL水样中加入10μL轮状病毒免疫磁珠、0.25μL Tween 20、孵育2 h可达到较好的分离效果;免疫磁珠可用于3%牛肉浸膏等常用病毒洗脱液中的轮状病毒的分离,表明该分离技术能与已有的病毒浓集方法良好地整合.免疫磁珠分离技术与实时定量PCR联合用于检测水中轮状病毒,全过程需时约5 h,检测限为1×10^4copies/mL（相当于3-4 PFU/mL）,检测结果与细胞病变试验检测结果有良好的线性相关关系（R^2=0.981 6）,表明其能较好地表征水样的病毒感染风险.对接种已知量的轮状病毒的污水处理厂二级出水、再生水、地表水、自来水等水样的实验表明,该法可用于各种水样中轮状病毒的检测.A quantitative and rapid detection method for rotavirus in water samples was developed, by using immunomagnetic separation combined with reverse transcription and real time polymerase chain reaction（IMS-RT-real time PCR）. Magnetic beads coated with antibodies directed against group A rotavirus were used to capture and purify the virus in water samples. The experimental results showed that IMS was optimized when 1 mL samples were supplemented with 10 μL of immunomagnetic beads, 2.5 μL of Tween 20 and incubated for 2 h. The IMS method was employed in the detection of rotavirus in seeded virus eluant such as 3 % beef extract successfully and thus manifested its compatibility with established virus concentration methods. The IMS-RT-real time PCR method could yield quantitative results within about 5 h with a detection limit at 1 × 10^4 copies/mL（equivalent to 3-4 PFU/mL）. The method exhibited a high level correlation（ R^2 = 0. 981 6） with cell culture assay, indicating that it could perform as well as cell culture assay does in infection tests. And the method functioned satisfactorily in seeded concentrate of secondary waste water treatment plant effluent, reclaimed water, surface water and tap water.

关 键 词：免疫磁珠分离前处理 实时定量PCR 轮状病毒 环境水样 

分 类 号：X832[环境科学与工程—环境工程]