不同稀释液对标准品核酸定量结果的影响研究

Study on the Effect of Different Dilutions on the Quantitative Results of Standard Nucleic Acids

对HBV 4th WHO IS 10/266(NIBSC)国际标准品稀释液进行选择和评估,筛选出稳定的对定值结果影响最小的稀释液,为PCR实验室标准品常规稀释提供理论基础和实验依据。通过选用不同浓度的稀释液,将国际标准品稀释成梯度,应用乙型肝炎病毒核酸检测试剂盒进行准确度和最低检测限验证。准确度验证每个稀释度重复检测6次;最低检测限验证每个稀释度重复检测20次;根据准确度和最低检测限的对比测试结果筛选出最优的稀释液,同时用筛选出的稀释液分别将企业参考品Q2稀释至线性范围内各浓度,每个浓度重复检测2次,以理论值为Y值检测值为X分别进行线性拟合(Y=aX+b)。结果:第一,10%小牛血清①和小牛血清②以及50%阴性血浆②和100%阴性血浆①稀释后检测结果与理论值最接近,△Log均小于0.1;第二,10%稀释度的小牛血清和阴性血浆稀释后的检测下限全部检出,50%稀释度的阴性血浆①和阴性血浆②符合95%的检出限,而原倍的只有阴性血浆①和1x TE全部检出。第三,对筛选后的稀释液稀释的企业参考品各浓度样本进行核酸定量检测,以理论值为Y值检测值为X分别进行线性拟合(Y=aX+b),结果显示a值均介于1.00±0.05之间,R2都大于0.98,线性关系良好,没有显著差异。通过实验数据的对比分析,10%小牛血清作为稀释液,对国际标准品定值影响最小稳定性最好,可作为实验室WHO标准品稀释液。

In this paper,the HBV 4th WHO IS 10/266(NIBSC)international standard diluent was selected and evaluated,and stable diluents that had the least impact on the determination results were screened out to provide theoretical and experimental basis for the routine dilution of PCR laboratory standards.By selecting diluents of different concentrations,the international standard is diluted into a gradient,and the hepatitis B virus nucleic acid detection kit is used to verify the accuracy and minimum detection limit.Accuracy verification Each dilution repeats the test 6 times;the minimum detection limit verifies each dilution repeats the test 20 times;According to the comparison test results of accuracy and the lowest detection limit,the optimal diluent was selected.At the same time,the company reference product Q2 was diluted to each concentration within the linear range with the selected diluent.Each concentration was tested twice,and use the theoretical value of Y and the test value of X to perform linear fitting(Y=ax+b).Results:First,the test results of 10%calf serum①and calf serum②,50%negative plasma②and 100%negative plasma①are the closest to the theoretical value after dilution,and△Log is less than 0.1;Second,the lower detection limit of 10%dilution of calf serum and negative plasma were all detected.The 50%dilution of negative plasma①and negative plasma②met the detection limit of 95%,while the original double only negative plasma①and 1x TE are all detected.Third,perform quantitative nucleic acid detection on samples of each concentration of the corporate reference product diluted with the diluent after screening,and perform linear fitting with the theoretical value of Y and the detection value of X respectively(Y=ax+b),and the results show that the a value is all between 1.00 and 0.05,R2 is greater than 0.98,the linear relationship is good,and there is no significant difference.Through comparative analysis of experimental data,10%calf serum is used as a diluent,which has the least influence on the value of international standards and has the best stability.It can be used as WHO standard diluent in laboratory.