实时荧光聚合酶链反应检测新型冠状病毒室内质控方法初探

Application on internal quality control system of SARS-CoV-2 detection by real time fluorescent polymerase chain reaction

目的绘制实时荧光聚合酶链反应(RT-PCR)定性室内质控图和基于循环阈值的Levey-Jennings质控图,探究新型冠状病毒RT-PCR检测的室内质控方法。方法使用无菌生理盐水按照1∶5、1∶10、1∶20、1∶30、1∶40对第三方质控物进行倍比稀释,确定室内弱阳性质控的稀释倍数。统计该实验室2022年1-2月新型冠状病毒RT-PCR阴阳性质控原始记录,以自定义数值绘制定性结果散点图。统计2022年1-2月每批次弱阳性质控的靶基因Ct值,分别对N基因和ORF1ab基因两组Ct值进行正态分布检验,根据前20次检测结果分别计算N基因和ORF1ab基因的靶值X和标准差s,绘制Levey-Jennings质控图。结果确定1∶30为每日室内质控的弱阳性质控品的稀释倍数。定性检测散点图可直观发现1次弱阳性质控检出阴性的“假阴性”反应。弱阳性质控N基因、ORF1ab基因Ct值均符合正态分布(P>0.05)。N基因Ct值靶值X为34.13,变异系数为1.91%,所有Ct值均在X±3 s以内,5次出现±2 s警告,符合Westgard质控规则。ORF1ab基因Ct值靶值为35.30,变异系数为2.58%,出现1次+3 s失控,5次±2 s警告,第31~41批次违反了10x规则,第73~80批次违反了R 4s规则。结论应用定性室内质控散点图和Levey-Jennings质控图建立的新型冠状病毒室内质控方法具有可行性,能够有效监测和预警检测过程中的质量问题,保证检测结果准确可靠。

Objective To explore a reasonable internal quality control system of SARS-CoV-2 nucleic acid detection by real-time PCR,real-time PCR qualitative testing quality control chart and Levey-Jennings quality control chart were draw.To explore the internal quality control method of real-time fluorescent polymerase chain reaction(RT-PCR)detection of SARS-CoV-2 by drawing qualitative internal quality control chart of RT-PCR and Levey-Jennings quality control chart based on cycle threshold.Methods Sterile normal saline was used to dilute the third-party quality control substance at the ratios of 1∶5,1∶10,1∶20,1∶30 and 1∶40,and then weakly positive quality control dilution ratio was confirmed.The original records of the negative and positive control of SARS-CoV-2 by RT-PCR from January to February 2022 in the laboratory were collected to draw a scatter plot of qualitative results with custom values.The Ct values of target genes in each batch from January to February 2022 were collected,and the Ct values of N gene and ORF1ab gene were tested for normal distribution,respectively.The target value(X)and standard deviation(s)of N gene and ORF1ab gene were calculated according to the results of the first 20 tests,and Levey-Jennings quality control chart was drawn.Results The dilution ratio of weakly positive quality control was 1∶30.Through the scatter diagram of qualitative results,the first“false negative”result could be found directly.The Ct values of N gene and ORF1ab gene obeyed a normal distribution(P>0.05).The target value of Ct value of N gene was 34.13,variable coefficient was 1.91%,all the Ct values were within X±3 s,and±2 s warning occured five times,which accorded with Westgard quality control rules.The target value of Ct value of ORF1ab gene was 35.30,variable coefficient was 2.58%,+3 s appeared once,±2 s warning appeared 5 times,lot No.31-41 broke the rule of 10x,and lot No.73-80 broke the rule of R 4s.Conclusion SARS-CoV-2 internal quality control system based on qualitative scatter chart and Levey-Jennings chart is feasible,which could effectively monitor and warn the quality problems in the process of detection,and ensure the accuracy and reliability of detection results.