荧光定量PCR法检测HCV-RNA质控物的制备及质控图的绘制

Preparation of internal quality control substance of real-time PCR to quantitative analysis of hepatitis C virus RNA and the quality control chart*

目的自制HCV-RNA荧光定量PCR室内质控物,利用Excel绘制质控图。方法取某一浓度HCV-RNA阳性血清,稀释至一定浓度,分装数管,-70℃保存。首次分两批(A组:第1批在最佳条件下检测;B组:第2批在常规条件下检测)连续检测20次,计算均值的对数值、标准差和变异系数,绘制常规条件下质控图,进行室内质控动态监测;第2次(C组:第3批在常规条件下检测)标本-70℃保存12月后,连续检测20次,计算均值的对数值、标准差和变异系数。结果 HCV-RNA室内质控常规条件下及标本-70℃保存12个月后,均值的对数值分别为5.023、5.041;标准差分别为0.228、0.231;变异系数分别为4.54%、4.58%;经两组均数对数的显著性t检验,均值的对数值之间无统计学差异(P>0.05);标准差、变异系数之间相差很小。结论荧光定量PCR检测HCV-RNA质控物的制备方法简单,性能稳定,质控图绘制方便,适合PCR实验室对HCV-RNA荧光定量的室内质控。

Objective To prepare an internal quality control substance of hepatitis C virus RNA（HCV-RNA） used in real time quantitative PCR,and prepare quality control chart with Microsoft Office Excel software. Methods HCV-RNA positive serums were prepared, subpackaged into several centrifuge tubes and saved at --70 ℃. For the first time, all samples were continuously de- tected for 20 times in two tranches（group A：the first batch was detected under optimal conditions; group B：the second batch was detected under routine conditions）. The logs of mean value（x）,standard deviation（s） and coefficient of variability（CV） were calcu- lated and quality control chart was plotted. For the second time, the serums were stored at --70 ℃ for 12 months（group C：the third batch was detected under routine conditions）. The logs of x,s and CV were calculated. Results The logs of x of HCV -RNA inter- nal quality control were 5. 023 and 5. 041,s were 0. 228 and 0. 231,and CV were 4.54% and 4.58%. The difference in the logs ofx had no significance（P〉0.05）. There was also no statistical difference of s and CV. Conclusion Internal quality control substance of HCV-RNA used in real-time quantitative PCR might be very stable and applicable in clinical laboratory,and quality control chart could be plotted easily.