新型冠状病毒核酸检测试剂盒中酶的性能比较研究  

Research on Functional Evaluation Method of Enzymes in Nucleic Acid Detection Kits

作　　者：杨镇州 李妍[1] 杨雪 刘刚[1] 梁文[1] YANG Zhenzhou;LI Yan;YANG Xue;LIU Gang;LIANG Wen(Laboratory of Biometrology,Chemical and Ionizing Radiation Metrolology Institute,Shanghai Institute of Measurement and Testing Technology,Shanghai 201203,China)

机构地区：[1]上海市计量测试技术研究院化学与电离辐射所生物计量实验室,上海201203

出　　处：《生物技术进展》2021年第6期777-782,共6页Current Biotechnology

基　　金：上海市计量测试技术研究院科研项目(H00RY2005)。

摘　　要：新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS⁃CoV⁃2)核酸检测试剂盒中酶的性能对检测效果有决定性作用。为探索逆转录酶和DNA聚合酶性能的快速评价方法,采用常用SARS⁃CoV⁃2核酸检测试剂盒,以SARS⁃CoV⁃2核酸标准物质为考察依据,对试剂盒中逆转录酶、DNA聚合酶的技术性能进行综合评价研究。第一步,通过分析cDNA产物与目标RNA浓度比值,考察逆转录酶的逆转录效率;第二步,以相同的cDNA为目标样品,进行实时荧光定量PCR分析;第三步,以新冠病毒RNA标准物质为目标样品,进行逆转录PCR(reverse transcription PCR,RT⁃PCR),比较试剂盒的扩增效率;第四步,考察试剂盒中酶的热稳定性。结果显示,所考察的试剂盒产品逆转录酶的逆转录效率为6.5%~80.7%,DNA聚合酶的扩增效率为90.4%~119.7%,而总体逆转录PCR的扩增效率为90.7%~102.4%。热稳定性评价结果显示,初始较好的酶活性可能在保存和运输期间发生质量变化。通过反映目前新冠病毒核酸检测试剂盒中酶的质量水平,以期对相关质控工作提供一定参考。The performance of enzyme in severe acute respiratory syndrome coronavirus 2(SARS⁃CoV⁃2)nucleic acid detection kit plays a decisive role in the detection effect.In order to explore the rapid evaluation method for the performance of reverse tran⁃scriptase and DNA polymerase,the technical properties of reverse transcriptase and DNA polymerase in the common SARS⁃CoV⁃2 nucleic acid detection kit were comprehensively evaluated based on the reference material of SARS⁃CoV⁃2 nucleic acid.In the first step,the reverse transcription efficiency of reverse transcriptase was investigated by analyzing the concentration ratio of cDNA product to target RNA.In the second step,the same cDNA was used as the target sample for real⁃time fluorescence quantitative PCR analysis to compare the amplification efficiency of DNA polymerase.In the third step,using COVID⁃19 RNA standard substance as target sample,reverse transcription PCR(RT⁃PCR)was used to compare the amplification efficiency of the kit.In the fourth step,to compared the amplification efficiency of DNA polymerase.The results showed that the reverse tran⁃scription efficiency of the tested kits ranged from 6.5%to 80.7%,the amplification efficiency of DNA polymerase ranged from 90.4%to 119.7%,and the amplification efficiency of the total reverse transcription PCR ranged from 90.7%to 102.4%.The thermal stability evaluation results showed that the enzyme activity,which was initially very good,may change in quality during storage and transportation.By reflecting the quality level of enzyme in SARS⁃CoV⁃2 nucleic acid detection kit,it has certain reference value for relevant quality control work.

关 键 词：新型冠状病毒 RT⁃PCR 逆转录 PCR效率 

分 类 号：R446[医药卫生—诊断学]