鼠疫耶尔森菌核酸检测试剂国家参考品的研制

Development of national reference of Yersinia pestis nucleic acid detection reagents

目的研制鼠疫耶尔森菌(以下简称鼠疫菌)核酸检测试剂国家参考品,以期用于鼠疫菌核酸检测试剂的评价和质量控制。方法选择5株鼠疫菌[EV株、0614F株、otten株、Tjiusidej(R)株、田鼠型201株]和10株阴性菌(3株小肠结肠炎耶尔森菌、牛种布鲁菌104M株、炭疽芽孢杆菌、伤寒沙门菌、土拉热弗朗西丝菌LVS株、假结核耶尔森菌、霍乱弧菌和志贺菌),经培养、收获、灭活,获得参考品原液,进行普通PCR扩增法验证后,制备成1套由5支阳性参考品、10支阴性参考品、1支最低检出限参考品(EV株阳性参考品经冻干制备而成)和1支重复性参考品(制备方法同最低检出限参考品)组成的国家参考品。采用荧光定量PCR法检测国家参考品的阴阳性符合率、均匀性和稳定性,并组织4家单位进行协作验证。结果阳性参考品原液经普通PCR扩增,均可见相应目的基因条带;阴性参考品原液未扩增出相应基因条带。荧光定量PCR法检测国家参考品阴阳性符合率均为100%。重复3次检测10支最低检出限参考品的3a基因Ct值差异无统计学意义(F=1.567,P=0.193)。与未经冻融的参考品比较,反复冻融3次的最低检出限参考品及阳性参考品3a基因Ct值差异无统计学意义(t分别为0.416和0.079,P均>0.05)。与未经高温保存的参考品比较,37℃放置5 d的最低检出限参考品Ct值差异有统计学意义(t=7.109,P=0.002),4和25℃条件下差异无统计学意义(t分别为0.341和0.751,P均>0.05);4、25、37℃放置5 d的阳性参考品Ct值差异均无统计学意义(t分别为2.442、0.373和-0.043,P均>0.05)。4家单位检测阳性和阴性参考品的符合率均为100%;2家单位检测最低检出限参考品的最低检出限为1×10^(2)个/mL,另2家为1×10^(3)个/mL;4家单位对重复性参考品检测结果的CV值<10.0%。结论本研究研制的鼠疫菌核酸检测试剂国家参考品具有良好的均匀性和冻融稳定性,于4和25℃下放置具有良好的加速稳定性,可用于鼠疫菌核酸检测试剂的评价和质量控制。

Objective To develop a national reference for nucleic acid detection reagents of Yersinia pestis,so as to use it for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents.Methods Five strains of Yersinia pestis[(EV strain,0614F strain,otten strain,Tjiusidej(R)strain and Microtus vole 201 strain)]and 10 strains of negative bacteria(3 Yersinia enterocolitica strains,Brucella bovis 104M strain,Bacillus anthracis,Salmonella typhi,Francis tulage LVS strain,Yersinia pseudotuberculosis,Vibrio cholerae and Shigella)were cultured,harvested and inactivated.The stock solution of the reference was obtained and verified by ordinary PCR amplification,and a set of national references composed of 5 positive references,10 negative references,1 reference of minimum detection limit(EV strain positive reference was prepared by freeze-drying)and 1 repetitive reference(the same preparation method as the minimum detection limit reference)was pre pared.Fluorescence quantitative PCR was used to detect the coincidence rate,uniformity and stability of the national reference products,and four laboratories were organized for collaborative calibration.Results After ordinary PCR amplification,the corresponding target gene bands were found in the stock solution of all the positive references,and no corresponding gene bands were amplified in the stock solution of negative references.The coincidence rates of the national references detected by fluorescence quantitative PCR were 100%.There was no significant difference in Ct values of 3a gene in 10 minimum detection limit references in three repeated detections(F=1.567,P=0.193).Compared with the references without freezing and thawing,there was no significant difference in Ct values of 3a gene of the minimum detection limit references and positive references after freezing and thawing for three times(t=0.416 and 0.079,respectively,each P>0.05).Compared with the references without high temperature preservation,the Ct value of the minimum detection limit reference stored at 37℃for 5 d was significantly different(t=7.109,P=0.002),while there was no significant difference at 4 and 25℃(t=0.341 and 0.751,respectively,each P>0.05).The Ct values of positive references had no significant difference after preservation at 4,25 and 37℃for 5 d(t=2.442,0.373 and-0.043,respectively,each P>0.05).The coincidence rates of positive and negative references in four laboratories were all 100%.The minimum detection limit of the minimum detection limit reference detected by two laboratories was 1×10^(2)/mL,and that by the other two laboratories was 1×10^(3)/mL.The CVs of the repetitive reference detection results in four laboratories were all less than 10.0%.Conclusion This set of national reference for Yersinia pestis nucleic acid detection reagents developed in this study has good uniformity and freeze-thaw stability,and has good accelerated stability at 4 and 25℃,which can be used for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents.