实验室病毒核酸检测工作中的实际问题与实验细节的改良

The Practical Issues of Nucleic Acid Testing Laboratory andImprovement of Experimental Protocol Details

目的针对病毒核酸检测多个环节,以近年来广泛流行的新型冠状病毒为例,设计模拟可能产生误差的实验,旨在探讨影响病毒核酸检测过程中的影响因素,帮助检验人员细化操作、改善实验流程以及提高工作质量。方法用不同量的液体石蜡封盖反应孔,分析其对反应结果的影响;设计不同的反应体系并扩增,分析预分装试剂误差与排枪使用误差对结果的影响;反应体系在不同温湿度环境放置不同的时长后扩增,分析环境和等待时长对结果的影响。结果上海伯杰检测试剂的最佳液体石蜡封盖量为20μL,中山达安和江苏硕士检测试剂的最佳液体石蜡封盖量为15μL;2倍分装试剂和排枪吸样误差不影响定性实验阴性样本和阳性对照品的结果判读;上海伯杰检测试剂较中山达安和江苏硕世试剂更稳定,冷藏不应超过4h。结论实验室在遵循实验室能力认可准则进行性能验证的基础上针对实际工作情况对实验细节进行测试和比较,了解不同试剂在本实验室温湿度环境下的扩增表现和稳定性,从而优化检测步骤,提高工作质量。

Objective To address potential simulated experimental errors in several aspects of the SARS-CoV-2 nucleic acid assay and to help testers refine the operation,improve the experimental process and enhance the quality of work.Methods The reaction wells were capped with different amounts of liquid paraffin to analyze its effect on the reaction results;Different reaction systems were designed to analyze the effect of errors in using multiple channel pipettes;The reaction systems were amplified after being placed in different temperature and humidity environments for different lengths of time,so as to evaluate potential impacts of environment and waiting time.Results The optimal liquid paraffin capping volume was 20μL for Shanghai Bojie assay and 15μL for Zhongshan Daan and Jiangsu Shuoshi assay;Multiple channel pipetting error did not affect the result interpretation of negative samples and positive control for qualitative experiments;Shanghai Bojie assay was more stable than Zhongshan Daan and Jiangsu Shuoshi reagents,and should not be refrigerated for more than 4h.Conclusion This study helps to optimize the nucleic acid testing procedure and to improve the quality of related lab work.