蚀斑法定量测定HSV-2病毒颗粒的优化实验条件  

Study on the optimization of plaque assay for the quantitative titration of HSV-2

作　　者：金晶[1] 类延花[1] 吴建军[1] 王明丽[1] 

机构地区：[1]安徽医科大学微生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2005年第4期321-323,共3页Acta Universitatis Medicinalis Anhui

基　　金：教育部科学技术研究重点项目(编号:01052);安徽省生物医药重大科技专项项目(编号:01303003)

摘　　要：目的建立稳定易操作的HSV-2蚀斑技术,为纯化和定量测定HSV-2毒株的感染性研究提供依据。方法HSV-2病毒悬液接种至Hela细胞单层,分别经37℃吸附和室温吸附60min,用含0·75%琼脂糖或0·75%羧甲基纤维素RPMI1640液覆盖,3d后用1%结晶紫染色5min,记数蚀斑数量,在挑取蚀斑接种扩增及保藏的同时,应用PCR法对蚀斑培养物进行鉴定。结果蚀斑经1%结晶紫染色在Hela细胞单层上呈紫色,多为圆形,着色深,散在分布,与周围区域界限明显。用琼脂糖和羧甲基纤维素覆盖下所形成的蚀斑大小及形状不同,但病毒滴度差异无显著性(P>0·05)。经室温和37℃吸附的病毒滴度差异亦无显著性(P>0·05)。结论建立了简便易行的蚀斑形成测定方法,可准确滴定HSV-2病毒感染数量和感染力。Objective To establish plaque assay for herpes simplex virus type 2. Methods HSV-2 was inoculated into Hela cells monolayer, adsorbed at 37℃ or at RT for 60 min, added one agar RPMI 1640 overlay or Methylcellulose RPMI 1640 overlay within 3 days and stained with 1% crystal iolet. Plaques were counted accurately. Plaques were removed with capillary pipettes before staining,inoculated and conserved. PCR was used to identify the HSV-2. Results Plaques were most of roundity, stained dark purple,separated on the Hela cells layer. Under agar overlay and Methylcellulose overlay, the samples formed plaques in different sizes and appearances, but titers had no significant difference （P 〉0.05）. After adsorption at RT and 37℃, the titers were no significant difference （ P 〉 0. 05 ） , but the cells grew better at 37℃. Conclusion Plaque assay is reliable and easy for titration HSV-2.

关 键 词：疱疹病毒2型 人 滴定分析法 

分 类 号：R373[医药卫生—病原生物学] R373.9[医药卫生—基础医学]