Research And Applications Of Recombinase Polymerase Amplification-based Point-of-care Testing Technologies For Pathogens

Molecular detection based on nucleic acid amplification is a popular trend of point-ofcare testing(POCT)technology.Although PCR amplification technology has been developed and mature,it is difficult to set up outside the laboratory due to its dependence on precise thermal cycling and supporting equipment,which cannot meet the demand of POCT.Recombinase polymerase amplification(RPA)is a kind of isothermal amplification technology,which has the advantages of low reaction temperature,strong amplification ability,high tolerance of samples and low equipment dependence.It is suitable for POCT.Based on RPA technology,this study conducted a series of POCT technology studies on pathogens aiming at the difficulties in multi-target detection,high-sensitivity and highspecificity detection,and detection of easily degradable RNA samples.The main work is as follows:The difficulty of RPA in multi-target detection is the interference of amplification preference to sensitivity.In this study,RPA and lateral flow strip(LFS)were combined to design and establish a duplex detection technology.Through systematic optimization of primer concentration ratio in the amplification system,the interference problem of amplification bias on sensitivity was solved,and the interference of non-specific interaction of LFS marker antigen and antibody on color signal was also solved.Two important foodborne pathogenic bacteria Vibrio cholerae and Vibrio vulnificus and two important aquatic diseases of shrimp acute hepatopancreatic necrosis disease(AHPND)and Enterocytozoon hepatopenaei(EHP)infection have been successfully realized,which contributes to the promotion of RPA technology for multiple and multi-target POCT detection.For high sensitivity and specificity,combining CRISPR technology with RPA can further improve them.But because CRISPR interferes with RPA amplification,the application of this combination of techniques for each specific test needs to be studied.In this study,a variety of technology combinations were designed for POCT detection of AHPND and EHP.One-tube detection for EHP is realized,and for AHPND the limit of detection was increased by an order of magnitude.POCT cases of CRISPR and RPA combined application were increased.SARS-Co V-2 nucleic acid tests are conducted on easily degradable RNA templates.Current methods require reverse transcription,which is prone to produce false negative results.In this study,the L/RPA SARS-Co V-2 detection technology was designed by ligase combined with RPA.The ligase reaction was used to replace reverse transcription,which can avoid false negative and has a limit of detection not lower than that of current detection methods.It provides a feasible choice for POCT detection of SARS-CoV-2.On this basis,CRISPR fluorescent molecular beacon and EXO fluorescent probe were combined to improve the detection stability.POCT detection of SARS-Co V-2 variants is conducive to rapid and targeted prevention and control.In this study,a new detection technique combining RPA,ligase and q PCR for SARS-Co V-2 variants was designed and established,which can effectively distinguish L452 R and E484 Q mutations on the Delta mutant.These studies have proposed a new idea of RNA template nucleic acid detection and promoted the application of RPA technology to the significant national demand for SARS-Co V-2 nucleic acid detection.In summary,in view of the difficulties in multi-target detection,high-sensitivity and high-specificity detection and detection of easily degradable RNA samples,a series of POCT detection technologies based on RPA were studied in this study,taking the detection of different types of pathogens as application models.It can help promote the wide application of this technology in POCT detection,promote the technological progress of POCT,and reserve more abundant tools to deal with the demand of POCT detection caused by pathogens.