| 崔亚婷，倪 军，马红玲，等.青蟹双顺反子病毒-1 原位杂交检测方法的建立与应用[J].广东农业科学,2015,42(11):130-134 |
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青蟹双顺反子病毒-1 原位杂交检测方法的建立与应用 |
| Development and application of an in situ hybridizationassay for detection of mud crab dicistrovirus-1 |
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| DOI： |
| 中文关键词: 拟穴青蟹 青蟹双顺反子病毒-1 地高辛核酸探针 原位杂交 |
| 英文关键词: Scylla paramamosain mud crab dicistrovirus-1 dig-labeled probe in situ hybridization |
| 基金项目: |
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| 摘要点击次数: 2040 |
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| 中文摘要: |
| ：根据青蟹双顺反子病毒-1（mud crab dicistrovirus-1，MCDV-1）基因的保守序列设计1 对引物，从感
染MCDV-1 的青蟹鳃组织中提取RNA 为模板进行RT-PCR 扩增，得到1 314 bp 的cDNA 片段。纯化后的PCR 产
物用地高辛（DIG）标记制备核酸探针，此探针与青蟹呼肠孤病毒（MCRV）、鲍肌肉萎缩症病毒（AbSV）、类疱疹
病毒（AbHV）、白斑综合症病毒（WSSV）、虎纹蛙病毒（TFV）、锦鲤疱疹病毒（KHV）等水生动物常见病毒均无
交叉反应，可以特异性的检测出MCDV-1，检出MCDV-1 的灵敏度为8.58×107 copies/μL。采用原位杂交检测方法
检测拟穴青蟹组织中MCDV-1 的组织分布，结果表明，拟穴青蟹中MCDV-1 RNA 的阳性信号主要分布于肝胰腺、
神经节、肠道和食管。 |
| 英文摘要: |
| Mud crab dicistrovirus-1 was isolated from massive deaths of mud crab and had a strong pathogenicity.Based on the MCDV-1 genome sequences，we designed a pair of primers and obtained a 1 314 bp fragment of MCDV-1gene by RT-PCR. Consequently，probe was prepared using DIG-labeling. The sensitivity and specificity of probe wereanalyzed by dot blot hybridization method. Results showed that this probe could detect a minimum of 8.58×107 copies/μLof MCDV-1 and was specific for MCDV-1 detection，without cross-reaction with MCRV（mud crab reovirus），AbSV（abalone shriveling syndrome-associated virus），AbHV（abalpone herpesvirus），WSSV（white spot syndrome virus），TFV（tigerfrogvirus）and KHV（Koi herpesvirus）. This probe was used to detect MCDV-1 in different tissues of diseasedmud crab by in situ hybridization and positive signals of MCDV-1 were mainly observed in hepatopancreas，ganglion，intestines and esophagus. |
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