利用原位聚合酶链反应对潜伏感染单纯疱疹病毒1型DNA在BALB/c小鼠神经元中的定位研究
Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction.
作者信息
Khansarinejad Behzad, Soleimanjahi Hoorieh, Ghaemi Amir, Tiraihi Taki, Pour Beiranvand Shahram
机构信息
Dept. of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Dept. of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, P. O. Box 14115-111, Tehran, Iran.
出版信息
Iran Biomed J. 2010 Jul;14(3):83-8.
BACKGROUND
Herpes simplex virus type-1 (HSV-1) establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3'-diaminobenzidine (DAB) substrate.
METHODS
Eight-week-old male BALB/c mice were inoculated via the eye by 104 plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR.
RESULTS AND CONCLUSION
The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia.
背景
单纯疱疹病毒1型（HSV-1）初次感染后在神经元中建立终身潜伏感染。基于地高辛配基-11-dUTP检测系统、抗地高辛配基过氧化物酶和3,3'-二氨基联苯胺（DAB）底物，采用直接原位PCR技术检测感染的BALB/c小鼠三叉神经节中潜伏的HSV-1 DNA的存在情况。
方法
8周龄雄性BALB/c小鼠经眼接种104个空斑形成单位的野生型伊朗HSV-1分离株。潜伏状态建立后，取出三叉神经节，采用原位PCR检测HSV-1基因组。最后，使用原位反应的扩增混合物作为常规凝胶基础PCR的模板，通过两轮PCR方法验证原位PCR的结果。
结果与结论
结果表明，使用过氧化物酶和DAB检测系统的直接原位PCR方法是检测潜伏感染神经节中潜伏HSV-1 DNA的有用手段。
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