Establishment And Application Of Nucleic Acid Detection Method Based On NASBA,Toehold Switch Sensor And Cell-free System

The outbreak of diseases has brought great disasters to human beings,and has become one of the greatest threats to human survival.Therefore,it is very important for human to detect diseases quickly,accurately and sensitively and get rapid and effective treatment.The common strategies of clinical diagnosis mainly depend on the culture and identification of pathogens,or the detection of specific antigens,antibodies or nucleic acids of pathogens.However,at present,conventional diagnostic methods,such as identification of microorganisms by observing the characteristics of culture,enzyme-linked immunosorbent assay and nucleic acid amplification detection,are usually carried out in medical laboratories.Their applications have been limited because of their common shortcomings,such as time-consuming,labor-consuming,high cost,expensive machine dependence and the inability to achieve rapid on-site detection.In recent years,with the increasing demand for on-site detection in clinical medicine,especially in resource poor areas,the point of care testing(POCT)technology has attracted more and more attention since it appeared,and the diagnosis technology based on biosensor is also developing.Therefore,it is necessary to develop new diagnostic methods to strengthen the timely understanding and intervention of these diseases.In this study,a cell-free chromogenic system based on?-galactosidase hydrolysis of chlorophenol red-?-D-galactopyranoside(CPRG)was established by reducing the volume of reaction and optimizing the composition and reaction conditions of the cell-free system.Then,a simple and low-cost method for the detection of pathogenic microorganisms was established by combining nuclear acid sequence-based amplification(NASBA)and toehold switch sensor.This method was verified by the detection of food borne pathogenic microorganisms.In this study,we first established a cell-free chromogenic system by optimizing the composition of cell-free system,which depends on?-galactosidase hydrolysis of CPRG.Rosetta(DE3)?lac Z extract was used in this system,no additional T7 RNA polymerase was added,only phosphoenolpyruvate was added to provide energy,and the optimal concentration of magnesium was 7 m M.By optimizing of reaction conditions of the cell-free chromogenic system,the reaction temperature range of cell-free detection system is wide,ranging from25°C-42°C.Under standing condition,the reaction time was 60 min.Different protectants can be selected to improve the stability of system under different storage conditions,such as adding 0.05 M trehalose at 4°C and 1%polyvinyl alcohol at-20°C.Based on the established cell-free chromogenic system,the stability of cell-free chromogenic system embedded in different types of filter paper was compared.These filter paper included quantitative filter paper with different pore size,qualitative filter paper with different pore size and whatman filter paper.It was found that the stability of cell-free chromogenic system embedded in rapid quantitative filter paper was the best.Using bioinformatics analysis and NUPACK software,15 and 25 toehold switch sensors were obtained for norovirus type GII.4 and norovirus type GII.17,respectively.Two toehold switch sensors,named GII.4-SN8 and GII.17-SN24,were obtained by screening in vivo and in vitro,and their specificity proved to be good.After NASBA reaction,only 500 f M target RNA was detected by GII.4-SN8 sensor,and 2.6 f M target RNA was detected by GII.17-SN24 sensor in the paper-based detection system.Therefore,a paper-based cell-free chromogenic system based on toehold switch sensor was established to detect and distinguish GII.4 and GII.17noroviruses.In order to improve the sensitivity of subsequent detection,NASBA system was optimized.The results showed that bovine serum albumin,dimethyl sulfoxide and sorbitol had no effect on the cell-free chromogenic system,and the optimal concentration of magnesium ion was 15 m M,the optimal addition of dimethyl sulfoxide was 1.5?L and the optimal amplification reaction time was 150 min in NASBA system.It was better to separate the mixture of cell lysate and magnesium ion from the other mixtures.Under the optimum concentration of magnesium ion,the addition of magnesium ion before and after freeze-drying had no effect on the overall color development.By NUPACK software analysis and the cell-free system screening,Ro V-S4,HAV-S3 and HAst V-S3 were finally obtained for rotavirus detection,hepatitis A virus detection and human astrocoronavirus detection,respectively,and they were highly specific.The sensitivity test results in 384-well plate showed that the lower visible limits of rotavirus,hepatitis A virus and human astrocoronavirus were 1.4 f M,14 a M and 20 f M,respectively.The above detection targets are RNA.In order to expand the range of detection target to DNA,a simple polymerase chain reaction with four cycles can be used to amplify the target.According to the specific genes of Listeria monocytogenes,Salmonella enteritidis,Staphylococcus aureus and Vibrio parahaemolyticus,four toehold switch sensors,named LM-S4,SE-S5,SA-S1 and VP-S1,were screened by NUPACK software and in vitro screening.The sensitivity test results showed that the lower visible limits were 10~5 CFU/m L,10~4 CFU/m L,10~6 CFU/m L and 10~6 CFU/m L for L.monocytogenes,S.enteritidis,S.aureus and V.parahaemolyticus,respectively.The results of specificity test showed that the toehold switch sensors of the four strains had good specificity.In conclusion,a detection platform based on isothermal amplification,toehold switch sensor and the cell-free system was established.This platform can detect the a M level of RNA and 10~4 CFU/m L of bacteria DNA without expensive instruments and professional operators,which provides a new idea for the development and application of POCT.