贝类派琴虫原位杂交检测方法的建立与应用  

Development and Application of an In-situ Hybridization Assay for Detection of Perkinsus spp. in Clams

作　　者：王彩霞[1] 冯春燕[1] 袁向芬[1] 邓俊花[1] 王宗祥[2] 吴绍强[1] 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100029 [2]内蒙古农业大学,呼和浩特010018

出　　处：《中国动物检疫》2014年第6期79-83,共5页China Animal Health Inspection

摘　　要：本研究选择派琴虫特异性引物PerkITS85和PerkITS750的PCR扩增产物进行地高辛标记制备探针,探索建立了派琴虫的组织切片原位杂交检测方法,并应用该方法检测我国黄海海域采集的贝类样品。研究结果表明,PerkITS85和PerkITS750的PCR扩增产物大小为673bp,利用随机引物扩增法制备的派琴虫地高辛标记探针特异性好,与包纳米虫和尼氏单孢子虫均无交叉反应。以世界动物卫生组织(OIE)推荐的瑞氏液体羟基乙酸盐(RFTM)培养法为金标准对本研究建立的原位杂交检测方法进行评价,其诊断敏感性(Dse)为90.9%,诊断特异性(Dsp)为100%。实际检测应用表明本研究所建立的派琴虫原位杂交检测方法是一种有效的派琴虫检测方法,可以应用于派琴虫病的检测。In this study,the conserved ITS fragment of Perkinsus was amplified by PCR using the genus-specific PerkITS85 and PerkITS750 as primers. The purified amplicons were digoxigenin-labeled and used as probes in the in-situ hybridizatio（SH）assay. Clam samples from the Yellow Sea were collected and screened by the developed ISH assay to confirm the perkinsus infection status. Results showed that a 673 bp fragment of perkinsus ITS gene was successfully amplified by PCR. The digoxigenin-labeled probe showed high specificity, without cross-reaction with Bonamia ostreae and Haplosporidium nelsoni and other aquatic parasites. The OIE recommended Ray＇s fluid thioglycollate medium（RFTM）culture assay was used as gold standard to evaluate the developed ISH assay,and it was found that the DSe of the ISH assay was 90.9%,and the DSp was 100%. Field application indicated that the developed ISH assay was an effective detection method for perkinsus and could be applied to perkinsus detection.

关 键 词：贝类 派琴虫 原位杂交 RFTM 检测 

分 类 号：S944.344[农业科学—水产养殖]