Denaturation Bubble-mediated Cascade Isothermal Nucleic Acid Amplification In A Single Closed Tube

In recent years,a variety of emerging infectious diseases have threatened people’s health around the world.The novel coronavirus(2019-ncov)alone has caused 218 million diagnoses.Nucleic acid detection has become an important method for clinical diagnosis of pathogens due to its excellent sensitivity and specificity.Among them,the isothermal amplification technology does not require the use of expensive thermal cycling equipment,which is very suitable for the requirements of point-of-care detection.However,the popular isothermal amplification systems in the market usually require multiple auxiliary proteins,enzymes or multiple primers,and the reaction system is relatively complex.Therefore,point-of-care detection methods with simple operation and good sensitivity are urgently needed.In contrast,denaturing bubble mediated Strand exchange amplification(SEA)only requires a pair of primers and a DNA polymerase to complete the reaction,which simplifies the reaction system,but it still has the problem of low sensitivity compared with other isothermal amplification techniques.In recent years,with the emergence of a variety of new cascade amplification technologies,it provides a good opportunity for the further development of SEA technology.Hyperbranched rolling circle amplification(HRCA)is often used in combination with other amplification methods due to its high sensitivity.Therefore,aiming at the short board of the low sensitivity of SEA technology,this study established a cascade isothermal amplification technology based on SEA combined with HRCA to amplify the amplification signal generated by SEA twice to improve the sensitivity of amplification detection,called Rolling cycle SEA(RC-SEA).The main contents of this study are as follows:Construction of RC-SEA method.In this study,a dumbbell DNA probe was designed and introduced into the SEA reaction.The dumbbell probe was used as the template for the double-stranded DNA amplicons produced in the first stage to undergo HRCA reaction to generate DNA products with tandem repeats,which achieved high sensitivity and specificity in a single tube.Detection of Staphylococcus aureus by RC-SEA method.RC-SEA could detect a minimum of 1.0×10~3 CFU/m L of S.aureus genomic DNA,and the results could be interpreted by fluorescence or colorimetric detection.Compared with SEA and the commonly used isothermal method Loop-mediated isothermal amplification(LAMP),The lowest detection limit of SEA was 1.0×10~6 CFU/m L,and the lowest detection limit of LAMP was 1.0×10~4 CFU/m L,indicating that RC-SEA had higher sensitivity than SEA and LAMP.Detection of SARS-CoV-2 RNA by RC-SEA method.Under optimal conditions,the detection limit of the assay for SARS-Co V-2 nucleocapsid protein(N gene)reached 10~3copies/m L.In addition,the performance of RC-SEA in colorimetric detection was investigated.Because the sensitivity of colorimetric dyes is lower than that of fluorescent dyes,the detection limit of RC-SEA based colorimetry is an order of magnitude higher than that of the related fluorescence method.It is demonstrated that this method has the potential to be used to construct a colorimetric detection platform.In conclusion,the RC-SEA cascade isothermal amplification method established in this study can be used for nucleic acid detection in a single tube under constant temperature.The method has the characteristics of simple system and high sensitivity,which has the potential for clinical pathogen detection and provides technical reference for the detection of emerging infectious diseases.