Study On The Detection Method Of SARS-CoV-2 Nucleic Acid Based On RTX(exo-) And PfAgo

The outbreak of COVID-19 poses a huge threat to people’s life,health and safety,and rapid and accurate detection of virus infection is particularly important for the control of the epidemic.Reverse transcription real-time fluorescence quantitative PCR(RT-q PCR)is currently the gold standard for detecting RNA viruses.This method requires first using reverse transcriptase to reverse transcribe viral RNA into c DNA,and then perform fluorescence quantitative PCR detection.Since the reverse transcriptase currently used is not heat-resistant,the entire detection process is divided into two steps: first perform reverse transcription reaction at a lower temperature,and then perform q PCR;if the RNA to be tested has a higher structure,it also needs to pretreat the sample,The operation is complicated and time-consuming.To solve this problem,this study used RTX(exo-)with high heat resistance and both reverse transcriptase activity and DNA polymerase activity instead of traditional reverse transcriptase,optimized reaction conditions to achieve RTX(exo-)single enzyme RT-PCR,and combined with PfAgo detection technology developed by our laboratory to develop a new SARS-CoV-2 detection method.The main results are as follows:1.According to the amino acid sequence of RTX(exo-)reported in the literature,16 site mutations were performed on KOD gene by Overlapping PCR to obtain target gene RTX(exo-),which was cloned into E.coli expression vector p ET23 a to obtain recombinant plasmid p ET23a-RTX(exo-).Recombinant protein RTX(exo-)with reverse transcriptase activity and polymerase activity was obtained through E.coli expression system.By optimizing RT-PCR amplification conditions,single enzyme RTX(exo-)was used to achieve ultra-fast RT-PCR for SARS-CoV-2 N gene target region,The time of RT-PCR was shortened to 25 min.2.By optimizing RT-PCR amplification conditions,single enzyme RTX(exo-)was used to achieve ultra-fast RT-PCR for SARS-CoV-2 N gene target region,Completed amplification within 25 min.By optimizing conditions,one-tube detection of SARS-CoV-2URPAND was achieved.Using paraffin wax to separate PfAgo reaction system from RT-PCR reaction system,One-tube detection was achieved.3.Through screening guide DNA that can efficiently mediate PfAgo cleavage,optimizing d NTPs concentration in RT-PCR reaction system,improved PfAgo enzyme cutting efficiency,completed cutting within 2-5 min.Combined with ultra-fast RT-PCR technology mediated by single enzyme RTX(exo-)developed a SARS-CoV-2 nucleic acid detection method based on RTX(exo-)and PfAgo(SARS-CoV-2 URPAND).This method can detect Ct value within 30 min new crown nucleic acid samples at 38.05.4、The one-tube method detection of SARS-CoV-2 URPAND was realized by optimizing the conditions.Use paraffin to separate the PfAgo reaction system and the RT-PCR reaction system,and complete the reaction in the same tube.In summary,this paper established a rapid and highly sensitive method for the detection of SARS-CoV-2 by using RTX(exo-),which is heat-resistant and has both reverse transcriptase and DNA polymerase activities,combined with PfAgo.Rapid detection of RNA viruses and even other RNA viruses provides a new strategy.