Rapid Detection Of Foodborne Pathogenic Bacteria In Liquid Environments Based On Isothermal Amplification And Lateral Flow Techniques

Simple,rapid and ultra-sensitive detection of foodborne biological pollutants in liquid environment is particularly important for pollutant control.Currently,the main detection methods applied to foodborne pathogens include traditional biological culture,antigen-antibody and nucleic acid detection methods.Although these methods have a certain degree of accuracy and specificity,they are time-consuming and require specialized operators,which seriously hinders their application in point-of-care testing(POCT).Therefore,it is particularly important to develop an efficient and rapid detection method that is simple and portable.In this work,a biosensor for rapid detection of foodborne pathogenic bacteria in liquid environments has been developed and deeply optimized.The sensor is based on lateral flow technology combined with recombinant enzyme polymerase isothermal amplification and can be applied to the immediate detection of foodborne pathogenic bacteria in the field.The main research work of this subject:(1)Salmonella enterica serotype Enteritidis(S.Enteritidis)is a widespread and highly infectious biological contaminant that causes zoonotic diseases.Most detection methods are qualitative,while quantitative methods are time-consuming and rely on large analytical instruments.Therefore,the development of a rapid,simple,and quantitative assay is important for the prevention and control of infection at the early stages of an outbreak.To address this requirement,we developed a lateral flow(LF)strip biosensor based on streptavidin-coated gold nanoparticles(Strept Av–Au NPs)combined with recombinase polymerase amplification(RPA)for the quantitative point-of-care testing of S.Enteritidis.Strept Av-Au NPs were simple to synthesise and bound firmly to DNA with end-modified biotin.RPA could greatly reduce detection time.A smartphone was used to collect images of the LF strip and input them into a laptop for quantitative analysis.The entire process was completed in less than 40 min and did not require large laboratory equipment or analytical instruments.For the synthetic S.Enteritidis DNA sequence and the cultured pure bacterial sample,the visual detection limits(LOD)were 1 × 10-11 M and2.5 × 103 CFU/m L,and the quantitative LOD were 5.5 × 10-13 M and 91.4 CFU/m L,respectively.In the reproducibility assay,RSD values of 5.88%,5.13%,and 4.45% were achieved for the three different concentrations of samples.This method has the advantages of low cost,high precision and rapid quantification,it is also suitable for the detection of various DNA-containing analytes.(2)Enterohemorrhagic Escherichia coli(EHEC O157: H7)is one of the most common biological contaminants causing diarrhea in humans,especially in children.EHEC O157: H7 can be transmitted through contaminated water or food,or through contact with animals or humans.Currently,the main methods used in the field of immunology to detect Salmonella are antigenic antibody assays and polymerase chain reaction-based nucleic acid assays,which require expensive costs and laboratory instrumentation and are not suitable for large-scale screening for pathogenic bacteria in healthcare-poor developing countries.To address this issue,a self-heating recombinase polymerase amplification-based integrated lateral flow strip biosensor was reported.The biosensor consists of lateral flow strip device,connector,RPA reaction chamber,support device and heating device,which is far less expensive to produce than the antigenantibody method and does not rely on any experimental apparatus.The entire process was completed in less than 20 min and did not require large laboratory equipment or analytical instruments.A visual limit of detection(LOD)of 1 × 103 CFU/m L,3.5 × 103 CFU/ml and a quantitative LOD of 104.02 CFU/m L and 366.70 CFU/m L were achieved for EHEC O157: H7 in pure samples in culture and EHEC O157: H7 in drinking water,respectively.In the reproducibility assay,RSD values of 5.39%,3.85%,and 4.82% were achieved for the three different concentrations of samples.The method not only allows rapid quantitative detection of Salmonella in samples,but is also cheap to produce,simple and portable,and has good potential for development in developing countries with poor medical facilities.(3)Immunological methods are effective means of detecting biological contaminants in the environment,such as antigen-antibody methods and enzyme-linked immunosorbent assays.Although they have high specificity and sensitivity,their limits of detection(LOD)are relatively large.Polymerase chain reaction(PCR)and its derivative methods(RT-PCR and multiplex PCR)perform well in terms of LOD,but they require specialized operators and expensive experimental equipment.To address this problem,a portable double signal amplification lateral flow strip biosensor was developed in this study for the ultrasensitive detection of bio-based contaminants in the water environment.The first amplification is the recombinase polymerase isothermal amplification(RPA)reaction,which can achieve exponential amplification of the target in a short time;the second amplification is the streptavidin-biotin system.The LOD of this method is much lower than that of the antigen-antibody method,while it is simple and portable without relying on too much experimental equipment,and is suitable for immediate detection in the field of drinking water.The lateral flow strip biosensor developed in this study has been successfully applied in the detection of S.Enteritidis and EHEC O157: H7 samples in drinking water,and the portability and detection limit of the biosensor have been continuously optimized,so that it can be better applied in the on-site detection of biological pollutants.At the same time,the biosensor is not only limited to the detection of foodborne biological pollutants,but also suitable for the detection of various contaminants containing DNA.