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目的建立狗肾细胞(MDCK)、人喉癌表皮细胞(Hep-2)和非洲绿猴肾细胞(Vero)共培养(下简称MHV细胞)分离单纯疱疹病毒(HSV)并用荧光PCR的CT值梯度参考曲线鉴定培养物的方法。方法选取1株某实验室保存的单纯疱疹病毒分离株,用培养液以5倍梯度倍比稀释成多个模拟样品。用此样品同时接种MHV细胞和Vero细胞,每个样品各接种5瓶,并设空白对照和无细胞对照。经37℃、8%CO2培养后,每隔12 h记录上述各组的细胞病变效应(CPE),培养在84 h后终止,所有培养物同批提取核酸,并用荧光PCR检测RNA。所有核酸同机检测,并记录CT值,无细胞对照组的CT值用软件制成梯度参考曲线,其余各组与梯度参考曲线对比。结果 MHV细胞和Vero细胞培养模拟样品后,均出现典型细胞病变,Vero细胞出现更晚,MHV组CT值低于Vero组和无细胞对照组(P<0.05),MHV组的CT值曲线位于梯度参考曲线的下方。结论 HSV病毒可以在MHV细胞内有效增殖,同一样品的CT值梯度参考曲线可用来客观评价该样品中病毒在细胞内的增殖。
OBJECTIVE: To isolate HSV from co-culture of canine kidney cells (MDCK), human laryngeal carcinoma epidermal cells (Hep-2) and Vero cells (Vero) Methods of identifying cultures with reference to curves. Methods A strain of herpes simplex virus isolated from a laboratory was selected and diluted into five or more simulated samples with a 5-fold gradient of culture medium. Simultaneous inoculation of MHV cells and Vero cells with this sample, each of which was inoculated with 5 vials, was set up with blank and cell-free controls. After incubation at 37 ° C and 8% CO2 for 12 h, the cytopathic effect (CPE) of each of the above groups was recorded, and the culture was terminated after 84 h. All cultures were subjected to the same batch of nucleic acid extraction and RNA detection by fluorescence PCR. All the nucleic acids were detected on the same machine and CT values were recorded. The CT value of the cell-free control group was graded by software and the rest of the groups were compared with the gradient reference curve. Results The typical cytopathic lesions were observed in MHV and Vero cells, Vero cells appeared later, the CT value of MHV group was lower than that of Vero group and no cell control group (P <0.05), and the curve of CT value in MHV group was located in the gradient Below the reference curve. Conclusion The HSV virus can effectively proliferate in MHV cells. The CT gradient reference curve of the same sample can be used to objectively evaluate the intracellular proliferation of the virus in this sample.