关键词: 新型冠状病毒, RT?PCR, 逆转录, PCR效率

Abstract:

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 transcriptase 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 transcription 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.

Key words: SARS?CoV?2, RT?PCR, reverse transcription, PCR amplification efficiency