自制HBV—DNA质控物的稳定性评价

Evaluation of the Stability of Self-made Quality Control Substances of HBV-DNA

目的制备HBV—DNA荧光定量PCR检测质控物，评价其稳定性。方法收集HBv_DNA阳性新鲜血清及HBV—DNA阴性新鲜血清，去除其他阳性指标（HAV—Ab，HCV-Ab，HIV—Ab和梅毒抗体）的标本后，分别充分混合，使用HBV-DNA阴性混合血清将HBv_DNA阳性混合血清稀释至一定浓度后，灭活、分装，随机分为5组，即A，B，C，D和E组。A组：立即在最佳条件下和常规条件下，应用HBV—DNA荧光实时定量PCR检测试剂盒分别检测20次；B组：放置4℃冰箱48h后检测；C组：放置4℃冰箱48h后，再置-20℃冷冻24h后复融检测；D组：在满足C组条件后，置-20℃冰箱冷冻24h后二次复融检测；E组：置-20℃保存12个月复融后检测。各组质控物均在常规条件下检测20次，分别计算各组均值的对数值、标准差和变异系数。结果A，B，C，D，E组HBV-DNA质控物均值的对数值分别为4．611，4．590，4．600，4．638和4．571，经方差分析F-2．32〈F0.05（4，95）2．47，P〉0．05，均值的对数值之间差异无统计学显著性意义；标准差分别为0．211，0．198，0．221，0．191和0．214；变异系数分别为4．5％，4．3％，4．8％，4．1％和4．6％，标准差、变异系数之间差异很小。结论HBV—DNA质控物制备简单，稳定性良好，适合临床检验中心进行室间质评（EQA）或现场EQA。

Objective To prepare quality control substances of HBV-DNA used in real-time quantitative polymerase chain re- action（real-time PCR） ,and observed the stability of it. Methods Collecting HBV-DNA positive and negative fresh serum of specimens. Specimens with positive indexes （HAV-Ab, HCV-Ab, HIV-Ab and syphilis antibody） were removed, then re- spectively mixed all the serums well. HBV-DNA positive mixed serums were diluted by using HBV-DNA negative mixed se- rums, then inactivated, s ubpackage, randomly divided into 5 groups, namely group A, group B, group C, group D and group E. Group A： immediately detecting the serums 20 runs respectively by diagnostic kit for quantification of quantification of HBV-DNA under optimal conditions and routine conditions. Group B： placed the serums in 4℃ refrigerator then detecting in 48 hours. Group C： placed the serums in 4℃ refrigerator for 48 hours, then in -20℃ for 24 hours then thawed. Group D： to meet the conditions of C group,in - 20℃ for 24 hours then thawed. Group E： in - 20℃ for 12 months then thawed. Each group was detected 20 runs respectively under the routine conditions. Logarithms of mean value of the groups, standards de- viation and coefficients of variation were calculated. Results The logarithms of mean value of the five groups were 4. 611, 4. 590,4. 600,4. 638 and 4. 571 respectively. By using analysis of variance,F=2.32〈F0.05 （4,95）2.47,P〉0.05,the differ- ence among the logarithms of the mean values had no significance. Standard deviations were 0.211,0. 198,0.221,0.191 and 0. 214 respectively. Coefficients of variability were respectively 4.5%, 4.3%, 4.8%, 4.1% and 4.6%. There was little difference among standard deviations, and coefficients of variability also. Conclusion Quality control substances of tfBV- DNA have simple preparation,good stability,and are suitable for external quality assessment （EQA） or spot EQA.