Veterinary and Agrochemical Research Centre, Department of Virology, Molecular Platform, Groeselenberg 99, B-1180 Brussels, Belgium.
J Virol Methods. 2010 Apr;165(1):76-82. doi: 10.1016/j.jviromet.2010.01.007. Epub 2010 Jan 29.
This study proposes a validation strategy for an automated extraction procedure, followed by RT-qPCR analysis. To avoid false-negative results, a triplex RT-qPCR was used which detects the target viral RNA, an internal and an external control. The methods to determine the validation parameters such as linearity, efficiency, analytical sensitivity, analytical specificity and intra- and interrun variability are described in detail. Special attention is given to the analytical sensitivity, which is determined by probit analysis. The limit of detection was set at the input concentration resulting in a positive result in 95% of the repeats. The intra- and interrun variability was analysed profoundly by testing samples covering a broad range of viral loads, from strong positive to weak positive. To increase the diagnostic capacity, the extraction protocol was automated with a JANUS Automated Workstation (PerkinElmer, Waltham, MA), which can extract 186 samples in 2h and 30 min. The automation of the extraction protocol implied some additional validation parameters to be determined such as position-effect, absence of cross-contamination and comparison with the manual protocol. These parameters give essential information about the performance of the robot and are of great importance when the automated assay is used in an accreditation system.
本研究提出了一种自动化提取程序的验证策略，随后进行 RT-qPCR 分析。为避免假阴性结果，使用三重 RT-qPCR 检测目标病毒 RNA、内部和外部对照。详细描述了确定验证参数的方法，如线性、效率、分析灵敏度、分析特异性以及内和间运行变异性。特别关注分析灵敏度，这是通过概率分析确定的。检测限设定为输入浓度，在 95%的重复中产生阳性结果。通过测试涵盖广泛病毒载量范围的样本（从强阳性到弱阳性），深入分析了内和间运行变异性。为了提高诊断能力，提取方案采用 JANUS 自动化工作站（PerkinElmer，Waltham，MA）自动化，该工作站可以在 2 小时 30 分钟内提取 186 个样本。提取方案的自动化需要确定一些额外的验证参数，例如位置效应、无交叉污染以及与手动方案的比较。这些参数提供了有关机器人性能的重要信息，当自动化检测在认证系统中使用时非常重要。
