Establishment Of Gene Editing Enzyme Cas12a-Mediated RPA Isothermal Amplification Nucleic Acid Detection System

An abnormal presence of nucleic acid in the body is often closely related to the infected virus or cancer disease.In order to treat such diseases quickly and effectively,in vitro nucleic acid testing must be carried out as soon as possible to identify abnormal nucleic acids.In addition,the influenza viruses have a wide-ranging impact on humans,especially influenza A viruses,so improving the detection efficiency of influenza A viruses is a long-term work.The rapid rise of in vitro nucleic acid detection methods in the past decade has been the use of Cas12a,Cas13a and other CRISPR-Cas effector proteins for rapid in vitro nucleic acid detection.However,due to the extremely small amount of nucleic acid in the sample,a certain technique is required to realize rapid nucleic acid amplification.I am committed to combining the LbCas12a fluorescence detection system with RPA amplification technology as the main technology for rapid in vitro nucleic acid detection to achieve detection of clinical samples of influenza A virus.In this paper,the preference of LbCasl2a attached cutting properties is firstly described,as well as the specificity and sensitivity of detection using only LbCasl2a system.Then explored the detection system combined with RPA amplification technology,the sensitivity increased by 6 orders of magnitude.Finally,a Nucleic acid detection system capable of detecting LbCasl2a combined with RPA amplification that can detect almost a single copy of the sample was established.I will continue to improve the system,and combine more modern technologies,such as lateral flow chromatography,direct observation of visible fluorescence,and microfluidics,to increase sensitivity and efficiency,reduce detection costs,improve the convenience of transportation and storage.In the end,I hope that,this system can be truly widely used for rapid nucleic acid detection in vitro.