直接逆转录原位PCR检测实验性汉坦病毒感染乳鼠脑组织中病毒RNA及其与原位分子杂交的比较  

DIRECT REVERSE TRANSCRIPTASE IN SITU POLYMERASE CHAIN REACTION FOR DETECTION OF VIRAL RNA IN THE BRAIN TISSUES FROM SUCKLING MICE EXPERIMENTALLY INFECTED WITH HANTAVIRUS: A COMPARISON WITH IN SITU HYBRIDIZATION

作　　者：杨守京[1] 刘彦仿[1] 贺宏印 刘俊彬[1] 

机构地区：[1]第四军医大学病理学教研室,西京医院传染科

出　　处：《中国组织化学与细胞化学杂志》1997年第4期29-36,共8页Chinese Journal of Histochemistry and Cytochemistry

摘　　要：出生后２～３ｄ的昆明种乳鼠，经腹腔接种１００个半数致死量的陈株汉坦病毒，每只０．０５ｍｌ，于接种后１、２、４、６、８、１０、１２、１４ｄ处死动物，每个时间点３～６只不等，取其脑组织固定于４％的多聚甲醛中，石蜡包埋制备５μｍ的连续组织切片，每时间点取１～２例组织切片，用逆转录原位ＰＣＲ（ＲＴ－ＩＳＰＣＲ）方法检测组织中病毒Ｓ片段ＲＮＡ，组织脱蜡后，经ＤＮａｓｅ、蛋白酶Ｋ、消化等予处理，用汉坦病毒ＲＮＡＳ片段特异的一对引物，在组织切片上进行病毒ＲＮＡ的逆转录和ＰＣＲ过程，直接将ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ掺入到扩增产物中，经过３０个ＰＣＲ循环后，用碱性磷酸酶标记的抗ｄｉｇｏｘｉｇｅｎｉｎ抗体免疫组化检测扩增产物，连续组织切片用ｄｉｇｏｘｉｇｅｎｉｎ标记的汉坦病毒Ｍ片段Ｇ２编码区ＲＴ－ＰＣＲ扩增产物的和Ｓ片段特异性探针进行原位分子杂交并与ＲＴ－ＩＳＰＣＲ结果进行比较，另外应用免疫组化检测该基因表达产物病毒核抗原（ＮＰ），结果，ＲＴ－ＩＳＰＣＲ在病毒感染１ｄ的乳鼠脑组织中检测到病毒ＲＮＡ扩增产物，扩增产物定位于神经细胞胞浆内，而原位分子杂交和免疫组化检测阴性。在病毒感染２ｄ及２ｄ以后的乳鼠脑组织中ＲＴ－Ｉ?Two to three day old new born suckling mice were intraperitonealy inoculated with 005 ml of 100LD50 of Chen strain hantavirus per mouse, and the mice with same volume of saline water as control. At 1, 2, 4, 6, 8, 10, 12, and 14 day after inoculation, 2 to 6 infected and control mice were sacrificed at each time point. The brains were taken out and fixed with 4% buffered paraformaldehyde for 24hrs, then embedded with paraffin and prepared for sections, 5um in thickness. Routinely Proteinase K and RNasefree DNase pretreatments were done. Direct reverse transcriptase in situ polymerase chain reaction (RTISPCR) method which combined intracellular reverse transcription (RT) and polymerase chain reaction (PCR) was employed using single primer pairs specific for hantaan virus S RNA. Target sequences of hantavirus S segment genes within hantanvirus infected cells in formalinfixed sections were amplified by in situ reveres transcription and then followed by 30 cycles of PCR. The amplificants were demonstrated by alkaline phosphataselabeled antidigoxigenin antibody and colored with a mixture of NBT and BCIP. The RTISPCR performed on the tissues either pretreated with DNase without RT or omission of primer or on the ISHverified viral RNA positive tissues were used to controls the protease conditions optimal both for DNase activity and for PCR and its specificity. The brains from uninfected animals were used as tissue negative controls. The distribution of hantavirus within brain tissues was also determined by in situ hybridization using digoxigeninlabeled probe specific for Hantaan S segment RNA and by digoxigeninlabeled RTPCR amplificants of G2 encoded gene of hantaan M RNA, and immunocytochemistry using monoclonal antibodies specific for hantavirus nucleocapsid protein respectively. In the brain tissues from mice within 24h inoculation, hantavirusinfected neuron exhibited cytoplasmic signal after RTISPCR, this was not seen in uninfected tissues or infected tissues with omissio

关 键 词：汉坦病毒 逆转录原位聚合酶链式反应 原位杂交 小鼠 脑 

分 类 号：R346[医药卫生—基础医学]