原位RT-PCR法检测柯萨奇B病毒RNA的实验研究

Experimental study on detection of viral RNA of Coxsackie virus B by means of in situ RT PCR

目的建立定位检测细胞或组织中柯萨奇Ｂ病毒ＲＮＡ的原位ＲＴ－ＰＣＲ方法（直接法）。方法对柯萨奇Ｂ病毒（１～６型）感染ＨｅＬａ细胞，经核酸酶（ＤＮａｓｅ和ＲＮａｓｅ）处理后的病毒感染ＨｅＬａ细胞以及非感染的ＨｅＬａ细胞进行原位ＲＴ－ＰＣＲ检测。结果经扩增后，病毒感染的ＨｅＬａ细胞呈蓝黑阳性信号；并且这种阳性信号经ＲＮａｓｅＡ作用而消失，却不受ＤＮａｓｅ作用的影响；而非感染的ＨｅＬａ细胞则不着色。结论原位ＲＴ－ＰＣＲ法可特异地检测柯萨奇Ｂ病毒感染ＨｅＬａ细胞中的病毒ＲＮＡ，是一种特异和敏感的定位检测方法。

Objective To establish a sensitive method for detecting Coxsackie virus B RNA in cells and tissues locationally.Methods HeLa cells infected with coxsackie virus B ,those treated by DNase,RNase and non viral infecting HeLa cells by nuclease treatment were detected.Results Non viral infecting HeLa cells were negative,virus infecting HeLa cells were stained with blue and black color,and the positive signals could be removed after digestion of RNase but not DNase.Conclusions It indicated that the method of in situ RT PCR is specific and sensitive,and will be useful for research on coxsackie virus B sustainable infection and related diseases.