反应体系混匀与否对新型冠状病毒实时荧光逆转录聚合酶链反应检测结果的影响

Influence of whether the reaction system is mixed or not on the detection results of SARS-CoV-2 by real-time fluorescent reverse transcription polymerase chain reaction

目的:探讨反应体系混匀与否对新型冠状病毒(SARS-CoV-2)实时荧光逆转录聚合酶链反应(RT-PCR)检测结果的影响。方法:两种不同逆转录时长的实时荧光PCR法试剂均按以下方法处理。试剂盒内自带的阳性对照用生理盐水配制成稀释度为1∶1,1∶10,1∶100三个浓度水平的试验样本。每个浓度水平的样本检测16次,其中8次的混匀体系先混匀再瞬离后上机检测(混匀组),而另外8次的反应体系则直接瞬离后上机检测(非混匀组),检测结果以阈值循环数(Ct值)表示。比较每个浓度水平混匀组与非混匀组的结果差异。当组间差异具有统计学意义时,则进一步分析差异有无临床意义(实际意义),当两组的均值之差在0.5以内,则认为差异无临床意义。结果:对于逆转录时间较长的试剂,各浓度水平组间差异均无统计学意义(P>0.05);对于逆转录时间较短的试剂,部分组间差异具有统计学意义(P<0.05),但无临床意义(组间均值之差在0.5以内)。结论:用实时荧光RT-PCR法检测新型冠状病毒核酸时,反应体系混匀与否对检测结果无影响。

Objective:To explore the effect of the results of SARS-CoV-2 detected by real-time fluorescent reverse transcription polymerase chain reaction(RT-PCR)between the reaction system mixed groups and non-mixed groups.Methods:Selected two RT-PCR reagents with different reverse transcription time,the positive controls of the reagent kits were diluted with normal saline into 3 concentration levels(1∶1,1∶10 and 1∶100).Divided into mixed groupsand non-mixed group according to the PCR reaction system was mixed or not,there were 8 wells for each group for every concentration level and then tested with the corresponding reagents.The differences of cycle threshold(Ct)between mixed groups and non-mixed groups were determined,and the data were statistically analyzed.When the difference was statistically significant,the deviation of the mean between the two groups was calculated,with the deviation less than 0.5 as the clinically acceptable standard.Results:For reagent with long time of reverse transcription,there were no statistically significant differences between mixed groups and non-mixed groups(P>0.05).For reagent with short time of reverse transcription,some differences of cycle threshold between mixed group and non-mixed group were statistical significance(P<0.05),but these differences were all less than 0.5,indicating good result consistency between the groups.Conclusion:When the SARS-CoV-2 nucleic acid was detected by real time RT-PCR method,there was no effect on the results between the reaction system mixed groups on non-mixed group.