摘要
目的建立在Vero细胞中检测Ⅰ型单纯疱疹病毒(herpes simplex virus-1,HSV-1)抗病毒药物敏感性的CCK-8(cell counting kit-8)法,经验证后,应用该方法进行野毒株及改造毒株的敏感性检测。方法优化筛选CCK-8试剂培养时间(3、4、5 h)、阿昔洛韦(acyclovir,ACV)稀释倍数(2、4倍)及样品培养时间(72、96、120 h)后,建立检测HSV-1抗病毒药物敏感性的CCK-8法。对建立的方法进行专属性、准确度、重复性、系统适用性验证后,用其检测野生型HSV-1及其改造毒株oHSV-GFP、oHSV-GM-CSF对ACV的敏感性,并与空斑法进行比较。结果建立的CCK-8法为:将HSV-1与不同浓度(5、1.25、0.313、0.0781、0.0195、0.00488μg/mL)ACV混合后接种Vero细胞,培养72 h后,加入CCK-8试剂,继续培养4 h后,采用酶标仪检测450 nm波长处的吸光度值,并计算ACV对HSV-1的半数抑制浓度(IC_(50))。测得ACV对oHSV-1标准毒株的IC_(50)值为0.13μg/mL。方法专属性、系统适应性良好,准确度验证样品回收率在70%~130%范围内,重复性验证样品回收率在70%~130%范围内,相对标准偏差(relative standard deviation,RSD)<20%。ACV对野生型HSV-1及其改造毒株oHSV-GFP、oHSV-GM-CSF的IC_(50)值分别为0.10、0.09、0.11μg/mL。空斑法测得的ACV对野生型HSV-1毒株的IC_(50)值为0.15μg/mL,与CCK-8法检测结果接近。结论建立的检测HSV-1对抗病毒药物敏感性的CCK-8法是一种简单、实用、有效的方法,可用于各种野生型HSV-1及改造毒株对各种抗病毒药物的敏感性研究。
Objective To develop a CCK-8(cell counting kit-8)assay for the determination of antiviral drug susceptibility of herpes simplex virus-1(HSV-1)in Vero cells,and to use the method for susceptibility determination of wild and modified strains after verification.Methods After optimizing and screening CCK-8 reagent culture time(3,4,5 h),acyclovir(ACV)dilution ratio(2,4 times)and sample culture time(72,96,120 h),the CCK-8 assay for the determination of antiviral drug susceptibility of HSV-1 was developed.The method was verified for specificity,accuracy,repeatability and system applicability,and then was used to detect the susceptibility of wild-type HSV-1 and its modified strains oHSV-GFP and oHSVGM-CSF to ACV,which was compared with plaque method.Results The developed CCK-8 method was as follows:HSV-1 was mixed with different concentrations(5,1.25,0.313,0.0781,0.0195,0.00488μg/mL)of ACV and then inoculated into Vero cells.After culturing for 72 h,CCK-8 reagent was added.After another 4 h of continuing culture,the absorbance value at 450 nm wavelength was detected by a microplate reader,and the IC_(50)of ACV on HSV-1 was calculated.The IC_(50)value of ACV against oHSV-1 standard strain was 0.13μg/mL.The method exhibited good specificity and system suitability.The sample recovery rates of accuracy and repeatability verification were both within the range of 70%to 130%,with the RSD<20%for repeatability verification.The IC_(50)values of ACV against wild-type HSV-1 and its modified strains oHSVGFP and oHSV-GM-CSF were 0.10,0.09 and 0.11μg/mL,respectively.The IC_(50)value of ACV against wild-type HSV-1 strain measured by plaque assay was 0.15μg/mL,which was close to that detected by CCK-8.Conclusion The developed CCK-8 assay for the determination of antiviral drug susceptibility of HSV-1 is a simple,practical and effective method,which can be used to study the susceptibility of various wild-type HSV-1 and its modified strains to various antiviral drugs.
作者
田纳
邓力
马天聪
李静华
高文蕊
于守智
潘淑媛
TIAN Na;DENG Li;MA Tiancong;LI Jinghua;GAO Wenrui;YU Shouzhi;PAN Shuyuan(Research and Development Office,Beijing Institute of Biological Products Co.,Ltd.,Beijing 100176,China)
出处
《中国生物制品学杂志》
2025年第11期1345-1350,共6页
Chinese Journal of Biologicals
基金
北京市高层次留学人才回国资助项目(49826532)。
