传染性造血器官坏死病毒焦磷酸测序检测技术的建立  

Development of Pyrosequencing Method for Detecting Infectious Hematopoietic Necrosis Virus

作　　者：尹伟力 刘瑶 倪杨帆 张四化 岳志芹[5] 孙涛[5] 

机构地区：[1]烟台出入境检验检疫局,山东烟台264000 [2]荣成出入境检验检疫局,山东荣成264300 [3]武汉市农业检测中心,湖北武汉430003 [4]武汉市动物疫病预防控制中心,湖北武汉430003 [5]山东出入境检验检疫局技术中心,山东青岛266000

出　　处：《动物医学进展》2017年第3期43-49,共7页Progress In Veterinary Medicine

基　　金：科技部"十二五"支撑计划项目(2013BAD12B02)

摘　　要：根据传染性造血器官坏死病毒(IHNV)基因序列,设计了IHNV最为保守的指纹序列,以及测序引物,建立了IHNV焦磷酸测序检测方法。对所构建的IHNV焦磷酸检测方法进行特异性试验和灵敏度检测。结果表明,所建立的方法特异性好,在8种鱼类病毒中能够特异性检测出目的病毒,检测方法灵敏度高,最低检出核酸量为10pg/μL。对建立的焦磷酸测序检测方法进行了实际应用研究,选取国内采集与进口的鱼类样本共计80批次进行IHNV检测。结果显示,焦磷酸测序检测方法可以有效的检出常规RTPCR不能检出的假阴性样本和弱阳性样本,该方法的灵敏度和特异性可以满足水生动物疫病检测的需要。By collecting the sequences of IHNV,the fingerprint sequences and the sequencing primers of the viruse were designed.The pyrosequencing method for detection of IHNV was established successfully.The method has good specificity and sensitivity,and can specifically identify the objective viruses in the eight fish viruses.The method has high sensitivity and the minimum detection limit of nucleic acid is 10pg/μL.The method was verified by the detection of IHNV in 80 batches of the samples collected from domestic and imported fishes.The results indicated that the pyrosequencing method can effectively detect the false negative and weakly positive samples,which the traditional method RT-PCR cannot detect.The specificity and sensitivity of the method met the requirement for detection of aquatic animal diseases.

关 键 词：传染性造血器官坏死病毒 焦磷酸测序 检测 

分 类 号：S854.43[农业科学—临床兽医学]