应用空斑形成法检测五种不同细胞对肠道病毒71型的敏感性

Detection of five different cells' sensitivities to enterovirus 71 using plaque forming assay

目的:建立肠道病毒71型(EV71)的空班形成方法,并比较BGM、VeroE6、MRC-5、HEp-2及A549五种细胞对EV71病毒的敏感性.方法:将不同稀释浓度的EV71病毒接种到五种细胞,分别覆盖三种不同的培养物,孵育一定时间后固定染色,比较出斑效果;以新建空斑形成法测定五种细胞感染EV7124-96h后培养液滴度,计算空班形成单位(pfu).结果:在五种细胞中,BGM和VeroE6覆盖琼脂糖凝胶的EV71可形成针尖样空班;覆盖1%甲基纤维素的EV71可形成直径大约1mm圆形或类圆形空班,形成空斑单位最多,病毒滴度分别达2.87×109pfu/L和1.65×109pfu/L;覆盖1.2%艾维素的EV71在BGM细胞中可形成直径大约0.5mm类圆形空斑,在VeroE6细胞中形成针尖样空斑;其他细胞均形成针尖样空斑.结论:空斑形成方法可对肠道病毒进行定量检测,BGM及VeroE6细胞为EV71的敏感细胞.

AIM： To establish the plaque assay for the quantitative detection of enterovirus 71 （EV71） and to compare sensitivity of buffalo green monkey （BGM） cells, veto E6 cells, MRC-5 cells, A549 and HEp-2 cells to it.METHODS： BGM cells and vero cells were transinfected with EV-71. Then the plates were covered with three kinds of overlay medium （i： 0.6% Agarose, ii： 1% methycellulose, iii： 1.2% Avicellulose） and incubated for 3 to 4 days. The plaques were stained, and the number of plaques was counted. The supernatants were harvested at 24-96 hours after infection for plaque assay, and this technique was used to measure the five cell lines simultaneously.RESULTS： The plaques appeared as clear cycles （range 0.5 to 1 mm） in BGM and vero E6 cells covered with 1% methycellulose and 1.2% avicellulose, whereas EV71 produced the pinpoint plaques covered with 0.6% agarose. The titer of vi- rus ranged from 1.65x109 pfu/L to 2.87x109 pfu/L.CONCLUSION： The techniques may benefit the quantitative detection of EV71. Furthermore, it indicates that BGM cells and vero E6 cells consti- tute a valuable and sensitive cell line system for enterovirus 71.