关键词: 新型冠状病毒, 实时荧光聚合酶链反应, 振荡混匀, 样本前处理, 假阴性

Abstract:

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 the first 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-time fluorescence 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 and flow 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<0.000 1）. 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<0.000 1）. 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.

Key words: Severe acute respiratory syndrome corona virus 2, Real-time fluorescence polymerase chain reaction, False negativity