分析仪器确认规范在实时荧光定量PCR仪确认中的实施

 目的  通过对实时荧光定量PCR（real-time fluorescent quantitative PCR，RTFQ-PCR）仪实施确认，了解分析仪器确认的具体步骤。方法  根据药品生产质量管理规范的要求，对该RTFQ-PCR仪分别进行安装确认、运行确认和性能确认，以及年度再确认。在性能确认中，通过定量PCR法和环介导恒温（loop-mediated isothermal amplification，LAMP）法检测分枝杆菌核酸来验证该仪器的准确性和重复性。结果  RTFQ-PCR仪的安装符合要求，各项测试操作均达到规定的标准。当采用定量PCR法检测TB进行RTFQ-PCR仪性能确认时，结核杆菌（tubercle bacillus，TB）核酸浓度的对数与循环阈值（cycle threshold，Ct）之间呈现良好的线性关系，相关系数绝对值达0.999 9；TB核酸回收率为97%～105%；对2个TB样品分别进行3次检测获得的Ct值的变异系数（CV）均小于5%。当采用LAMP法检测分枝杆菌进行RTFQ-PCR仪性能确认和再确认时，对分枝杆菌阳性对照和样品分别进行3次检测获得的Ct值的CV均小于5%。结论  该RTFQ-PCR仪得到成功确认，各方面均符合规定的要求。

Implementation of analytical instrument qualification in the real-time fluorescent quantitative PCR system

 Objective  To understand the specific contents of analytical instrument qualification by implementing qualification of the real-time fluorescent quantitative PCR (RTFQ-PCR) system.  Methods  According to the requirement of GMP, installation qualification, operational qualification, performance qualification (PQ) and requalification of the RTFQ-PCR system were implemented. In PQ, accuracy and repeatability of the RTFQ-PCR system were validated by detecting mycobacteria DNA with quantitative PCR method and loop-mediated isothermal amplification (LAMP) method.  Results  The RTFQ-PCR system installation met the requirement, and each testing operation reached the required standard. When PQ of the RTFQ-PCR system was implemented by quantitative PCR detection, there was a good linear relationship between the logarithm of tubercle bacillus (TB) DNA concentration and cycle threshold (Ct), and the absolute value of correlation coefficient reached 0.999 9. Recoveries of TB DNA were 97%~105%. Coefficient variations (CVs) of Cts obtained by detecting two TB samples for three times each with quantitative PCR method were both less than 5%. When PQ and requalification of the RTFQ-PCR system were implemented by LAMP detection, CVs of Cts obtained by detecting the positive control and the sample of mycobacteria for three times each with LAMP method were all less than 5%.  Conclusion  Qualification of the RTFQ-PCR system is implemented successfully, and all aspects of the RTFQ-PCR system meet requirement of analytical instrument qualification.