| H9N2亚型禽流感病毒原位杂交检测方法的建立 |
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| 引用本文： | 张万坡,程国富,谷长勤,李德学,宋念华,毕丁仁,胡薛英,胡思顺,胡智斌.H9N2亚型禽流感病毒原位杂交检测方法的建立[J].中国兽医学报,2005,25(4):371-373. |
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| 作者姓名： | 张万坡 程国富 谷长勤 李德学 宋念华 毕丁仁 胡薛英 胡思顺 胡智斌 |
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| 作者单位： | 1. 华中农业大学,动物医学院,湖北,武汉,430070
2. 湖北省动物疫病诊断中心,湖北,武汉,430064 |
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| 基金项目： | 湖北省科技攻关项目(2004AA202B04) |
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| 摘 要： | 根据已知禽流感病毒(Avian influenza virus，AIV)核蛋白(NP)基因序列，选取其中保守区域设计出1时引物。以RT-PR方法扩增出543bp的NP基因片段，采用地高辛标记制备探针。MDCK细胞人工感染H9N2亚型禽流感病毒后，不同时间取样制备细胞涂片，进行原位杂交，研究了禽流感病毒时MDCK细胞的感染。探讨了原位杂交的条件及其应用于检测AIV感染的可行性。结果显示，感染24h后，原位杂交可检测出细胞内的病毒RNA，并且杂交信号的强度与感染时间的长短有关。研究表明。原位杂交检测禽流感病毒，不但能很好地表现病毒和细胞的位置关系,而且具有较好的特异性和灵敏性，为禽流感病毒的组织原位杂交检测奠定了实验基础。
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| 关 键 词： | 原位杂交 禽流感病毒 MDCK细胞 |
| 文章编号： | 1005-4545(2005)04-0371-03 |
| 修稿时间： | 2004年3月1日 |
Detection of H9N2 Subtype Avian Influenza Virus by in Situ Hybridization |
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| ZHANG Wan-po,CHENG Guo-fu,GU Chang-qin,LI De-xue,SONG Nian-hua,BI Ding-reng,HU Xue-ying,HU Si-shun,HU Zhi-bin.Detection of H9N2 Subtype Avian Influenza Virus by in Situ Hybridization[J].Chinese Journal of Veterinary Science,2005,25(4):371-373. |
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| Authors: | ZHANG Wan-po CHENG Guo-fu GU Chang-qin LI De-xue SONG Nian-hua BI Ding-reng HU Xue-ying HU Si-shun HU Zhi-bin |
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| Abstract: | In situ hybridization(ISH) is a technology for the localization of DNA and RNA sequence in tissues and cells.A sensitive and specific method of detection of AIV in MDCK cells is described here.Based on the AIV NP gene sequence,we designed a pair of primers and obtained a 543 bp fragment of NP gene by RT-PCR.Consequently,probe is prepared using DIG-labeling.MDCK cells infected by H9N2 AIV were harvested and spread on slides.After a series of process of fixation,proteinase K digestion and so on,the specimens on slides were then hybridized with the probe,followed by color reaction.The results showed that,after 24 h of inoculation,a positive hybridization signal could be detected in the cell.And the intensity of the signal is consisted with time of infection.However,no positive signal can be seen in the normal cells.It can be concluded that,ISH has not only the feasibility of in situ identification of cells infected by AIV,but also a relative high sensitivity. |
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| Keywords: | in situ hybridization(ISH) avian influenza virus(AIV) MDCK cell |
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