新型冠状病毒核酸检测7种振荡混匀方法效果评价

Evaluation of 7 oscillating mixing methods for SARS-CoV-2 nucleic acid determination

目的对新型冠状病毒(SARS-CoV-2)核酸检测7种振荡混匀方法进行比较,确定有效方法的最优参数。方法分2个阶段进行研究:第1阶段,采用小样本(每种方法30例),从形态学、流式细胞术、实时荧光聚合酶链反应(PCR)3个层面评价7种振荡方法的有效性,并确定最优时间参数;第2阶段,采用大样本(2150例),验证最优参数下多管涡旋和超声振荡的效果。结果形态学和流式细胞术分析结果显示,除超声振荡外,其他6种振荡混匀方法均会导致保存液中上皮细胞明显增多(P<0.05)。实时荧光PCR结果显示,颠倒和回旋振荡处理后,核酸扩增未见内参循环阈值(Ct)明显下降(P>0.05);吹打6次和往复振荡5 min处理后,核酸扩增未见内参Ct值下降;单管/多管涡旋和超声振荡处理后,核酸扩增内参Ct值显著下降(P<0.0001)。大样本量实时荧光PCR结果显示,多管涡旋振荡2 min较超声振荡5 min效果更优(P<0.0001)。结论SARS-CoV-2核酸检测采用2min涡旋振荡混匀的方法进行前处理可提高检测效率。

Objective To evaluate the effect of 7 oscillating mixing methods for severe acute respiratory syndrome corona virus 2(SARS-CoV-2)nucleic acid determination and determine the optimal parameters of effective method.Methods In thefirst stage,30 samples(small sample)of each oscillating mixing method were used to evaluate effectiveness and determine the optimal time parameters from morphology,flow cytometry and real-timefluorescence polymerase chain reaction(PCR).In the second stage,large sample was used to verify the effectiveness of multi-tube vortex treatment and ultrasonic shock under the optimal parameters,and the oscillation effects of multi-tube vortex treatment and ultrasonic waves were compared.Results Morphology andflow cytometry results showed that epithelial cells in preservation solution were increased by 6 oscillating mixing methods,except ultrasound(P<0.05).Real-time fluorescence PCR showed that there was no decrease in internal reference cycle threshold(Ct)in nucleic acid amplification after reverse and cyclotron shock treatments(P>0.05).There was no decrease in internal reference Ct in nucleic acid amplification after blowing for 6 times and reciprocating shock for 5 min.The internal reference Ct in nucleic acid amplification after single/multi-tube vortex treatment and ultrasonic shock was decreased(P<0.0001).Real-time fluorescence PCR results of large sample further verified the effectiveness of multi-tube vortex treatment for 2 min and ultrasonic shock for 5 min(P<0.0001).Conclusions For the pre-treatment of SARS-CoV-2 nucleic acid samples,2 min vortex oscillating mixing method can be adopted,which is simple and easy to use and has good oscillation effect.