免疫磁珠富集与SYB RGreen荧光定量PCR技术联合检测水体中甲肝病毒  

DETECTION ON HEPATITIS A VIRUS IN WATER SAMPLE USING IMMUNOMAGNETIC BEADS-BASED CONCENTRATION COMBINED WITH SYBR GREEN REAL-TIME PCR

作　　者：乔煜婷[1] 曹华斌[1] 胡国良[1] 陈全姣[2] 

机构地区：[1]江西农业大学动物群发性疾病监测和防治研究所,南昌330045 [2]中国科学院武汉病毒研究所,武汉430071

出　　处：《内蒙古农业大学学报（自然科学版）》2014年第2期13-18,共6页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：国家科技支撑计划(2013BAK12B05)

摘　　要：本实验旨在建立一种免疫磁珠与SYBR Green荧光定量PCR技术联合检测水体中甲肝病毒的方法。通过制备特异性甲肝病毒免疫磁珠,优化病毒核酸提取方法,建立了免疫磁珠富集病毒和荧光定量PCR检测水体中甲肝病毒的方法。研究结果显示,1mg免疫磁珠与75μg甲肝病毒单克隆抗体偶联时,有最大抗体偶联量;该偶联后的磁珠富集甲肝病毒效率为92.87%。研究结果还显示,SYBR Green荧光定量PCR检测的标准曲线线性关系良好(R^2=0.999),检测极限低至9.27×10~1拷贝/μL。本研究结果表明,免疫磁珠与SYBR Green荧光定量PCR技术结合,具有特异性强、灵敏度高、可信度强、简便快捷的优点,本方法对水体中甲肝病毒污染状况检测与评价提供了有力的技术支持。The aim of the experiment was to build up a detection method of hepatitis A virus in water sample,by using immunomagnetic beads- based and SYBR Green real- time PCR（IMB- real- time PCR）.By preparing the magnetic beads coated with HAV monoclonal antibodies and optimizing the viral RNA extraction methods,the detection methods of hepatitis A virus in water sample were set up,such as utilizing immunomagnetic beads- based virus concentration and real- time PCR.The data showed that 1mg immunomagnetic beads which coated with 75μg HAV antibodies had the greatest efficiency（92.87%） to capture HAV in water sample.Meanwhile,the standard curve of real- time PCR had good linear relationship（reliability was 0.999）.The limit of detection was improved as low as 9.27×10^1 copies/μL.The results indicated that the immunomagnetic beads- based with SYBR Green real- time PCR is a rapid,sensitive,specific and reliable method,which provides a strong technical basis for detection of HAV and evaluation in water.

关 键 词：免疫磁珠 荧光定量PCR 甲肝病毒 

分 类 号：S855.99[农业科学—临床兽医学] R373.2[农业科学—兽医学]