安徽省首例新型冠状病毒的分离培养与TCID50测定

Isolation and identification of the first strain of SARS-CoV-2 in Anhui province and measuring the TCID50

目的建立新型冠状病毒分离和培养方法,为抗新型冠状病毒药物研发和动物实验打下理论基础并提供实验依据,同时探究病毒TCID50与基因拷贝数之间可能存在的数量关系。方法采集本省一例新型冠状病毒感染的肺炎患者的咽拭子标本,将处理后的标本接种于Vero细胞并进行病毒分离,通过qPCR和二代测序鉴定该分离毒株,同时测定该毒株的半数组织细胞感染量(50%tissue culture infection dose,TCID50)。稀释质粒标准品并进行qPCR,建立标准品线性回归曲线,检测不同代次、不同TCID50的病毒基因拷贝数。结果将标本接种于Vero细胞后,24~72 h未发现明显细胞病变效应(Cytopathogenic effect,CPE),96 h细胞出现明显CPE,可判定为+,120 h细胞全部发生病变。结论新型冠状病毒可在Vero细胞系中增殖,并可连续传代;在限定病毒传代次数等条件下,病毒TCID50和病毒基因拷贝数之间存在对应的数量关系,可实现二者间的替换。

Objective To establish the cell culture and isolation methods of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)to provide experimental basis for the anti-SARS-CoV-2 drug development and animal experiments,and to explore the possible quantitative relationship between the 50%tissue culture infection dose(TCID50)of the virus and the number of gene copies.Methods The throat swab sample was collected from a COVID-19 patient in our province.The sample was inoculated into the Vero cell line after pre-treatment to isolate the SARS-CoV-2.The isolated strain was identified by qPCR and second generation sequencing,and the TCID50 was also measured.The plasmid standard was diluted and tested by qPCR.The standard curve was established to detect the number of gene copies of the virus of different passages and TCID50.Results No significant cytopathogenic effect(CPE)was observed from 24 to 72 h after inoculation in the Vero cells.CPEs were found from 96 h and was judged as"+".After 120 h,all cell cultures showed CPE.Conclusions The SARS-CoV-2 can be proliferated and cultured continuously in Vero cell line.Under the conditions of limited virus passages,a certain quantitative relationship between the TCID50 and the number of copies of virus genes was found and can be used to convert both criteria.