金黄色葡萄球菌核酸检测试剂盒用国家参考品的研制

Development of national reference for Staphylococcus aureus nucleic acid detection kit

目的研制金黄色葡萄球菌核酸检测试剂盒用国家参考品。方法将10株金黄色葡萄球菌和10株阴性对照代表性菌株分别在适宜条件下复苏培养,制备合适浓度的菌悬液,并加热灭活和分装。随机抽样不同候选参考品样品10支进行均匀性分析,并将样品分别置于2~8、25、37℃条件下保藏以及反复冻融,评价标准物质的稳定性。同时对候选参考品的准确性、特异性、重复性、最低检出限进行分析,并组织8家实验室进行协作标定。结果制备了由阳性、阴性、重复性和最低检出限样品组成的金黄色葡萄球菌核酸检测试剂盒用候选参考品。不同候选参考品不同样品间的Ct值的变异系数在1.23%~3.86%之间,表明制备候选参考品均匀性良好。同时证实P1~10阳性和最低检出限候选参考品样品在反复冻融及加速破坏条件下稳定性良好。应用3个不同厂家生产试剂盒进行重复性验证实验显示,样本的Ct值均小于5.0%,表明候选参考品的重复性良好。除P6阳性候选参考品样品不能被部分试剂盒检出,其余阳性参考品样品均能成功检测、最低检出限为1.0×10^(3)个/m L。协作标定结果进一步表明阳性、阴性、重复性和最低检出限候选参考品均符合国家参考品的各项要求。结论研制的金黄色葡萄球菌核酸检测试剂盒用国家参考品已获得批准,可用于金黄色葡萄球菌核酸检测试剂盒的质量控制和评价。

Objective To develop national reference for Staphylococcus aureus nucleic acid detection kit.Methods The selected 10 strains of S.aureus and 10 strains of non-S.aureus were cultured in the suitable media,respectively,the bacterial suspensions with a suitable concentration were prepared,of which were inactivated by heating and distributed.Ten different candidate reference samples were randomly selected for homogeneity analysis,and the samples were stored at 2-8,25 and 37℃as well as repeatedly freeze-thaw to evaluate the stability of the standard materials.Furthermore,the accuracy,specificity,repeatability,and lowest detection limit of candidate reference samples were analyzed,and 8 laboratories were organized to conduct collaborative calibration.Results The national references for S.aureus nucleic acid detection kit were successfully developed,which comprised of positive,negative samples,minimum detection limit and precision components.The coefficients of variation of the Ct values between different candidate reference samples were between 1.23%and 3.86%,indicating that the uniformity of prepared candidate reference samples was good.Furthermore,it was confirmed that the P1-10 positive and lowest detection limit candidate reference samples had good stabilities under repeated freezing and thawing and accelerated destruction conditions.Ct values of the samples determined by using kits produced by 3 different manufacturers were all less than 5.0%,suggesting that the candidate reference products had good reproducibility.Except for the P6 positive candidate reference sample that could not be detected by some kits,the rest of positive reference samples could be successfully detected,the minimum detection limit was 1.0×10^(3)CFU/mL.The collaboration results further indicated that the positive,negative samples,minimum detection limit and precision components in candidate references conformed to the requirements of national reference product.Conclusion The national reference for S.aureus nucleic acid detection kit developed has been approved,which can be used for the quality control and evaluation of S.aureus nucleic acid detection kits.