Combination Of Immunomagnetic Separation And Loop-mediated Isothermal Amplification(IMS-LAMP) For Rapid Detection Of Three Foodborne Pathogens

Food-borne pathogens are an important factor causing food safety problems in today's society.Staphylococcus aureus,Salmonella and Escherichia coli O157:H7 are three common food-borne pathogens in food,which can cause diarrhea and vomiting in humans.It can even cause death in severe cases.The shortcomings of the current commonly used food-borne pathogen detection technology are long detection period,expensive equipment and reagents,complicated operation process,and complicated and cumbersome sample preparation.Immunomagnetic separation(IMS)can quickly isolate pathogenic bacteria,protect the biological activity of the isolated bacteria,effectively eliminate substances that affect nucleic acid amplification,and improve the sensitivity of detection.The process is simple and easy to use and does not require expensive centrifuge equipment.It is a pretreatment method with the greatest potential to concentrate and isolate pathogenic bacteria.Loop-mediated isothermal amplification(LAMP)detection technology is a molecular biological method suitable for grass-roots detection of specific pathogenic bacteria.It has high accuracy,short detection time,low requirements for instruments and equipment,and is suitable for rapid detection pathogenic bacteria,but there are many interfering substances in food,such as protein,which will affect the sensitivity of LAMP detection.Appropriate methods are needed to quickly enrich pathogenic bacteria in food and eliminate the impact of food matrix.This study combines the immunomagnetic bead enrichment method and the loop-mediated isothermal amplification detection technology to establish a rapid detection scheme for the pathogenic bacteria IMS-LAMP,which is a simple,fast,and highly specific gene detection technology,suitable for testing in the field of food contamination.The main research contents and conclusions are as follows:1.Construction of immunomagnetic bead enrichment methods for S.aureus,Salmonella and E.coli O157:H7.The biotin-labeled Salmonella antibody,S.aureus protein antibody,and streptavidin magnetic beads were conjugated,and the amount of1mg nanomagnetic conjugate antibody was 10?g and 6?g,respectively.The enrichment conditions of Salmonella immunomagnetic beads,S.aureus immunomagnetic beads,and E.coli O157:H7 immunomagnetic beads were optimized.The addition amount of immunomagnetic beads in the 1 m L reaction system was 400?L,300?L,and 60?L,respectively.The reaction times were 45 min,30 min and 30min,respectively.The immunomagnetic beads of the three pathogenic bacteria had good specificity,no capture phenomenon for five non-target bacteria and high sensitivity,and still could capture when the concentration of the bacterial solution was as low as 10~1 CFU/m L.2.Establishment of LAMP detection method for S.aureus,Salmonella and E.coli O157:H7.A fluorescent dye was added to the LAMP reaction solution to establish a real-time fluorescent LAMP method.Primers were designed based on the conserved sequences of Salmonella inv A gene,S.aureus nuc gene and rfb E gene of E.coli O157:H7,and primer selection was performed to select the best primers inv A-2,nuc-1 and rfb E-1.The optimization experiment was carried out to influence the temperature of LAMP reaction,and the optimal reaction temperature was determined to be 63?.The specificity of the LAMP reaction of the three pathogenic bacteria is good,and there was no amplification reaction to the five non-target nucleic acids.Negative repeat results were good,and no amplification reaction occurred in 20negative repeat tests.The LAMP method had high precision.For target plasmids with a concentration of 10 pg/?L and 1 pg/?L,the CV values of the LAMP method for Salmonella plasmids are 3.58%and 3.22%,and the CV values for S.aureus plasmids were 3.65%and 3.47,the CV values for E.coli O157:H7 plasmid were 3.40%and3.28%,respectively.The sensitivity was high.The sensitivity of Salmonella plasmid was 1.91×10~2 copies/?L,the sensitivity of S.aureus plasmid was 2.72×10~2 copies/?L,and the sensitivity of E.coli O157:H7 plasmid was 2.28×10~2 copies/?L,which was the same as that of the control Real-time PCR.The detection of different concentrations of pure culture bacteria,the sensitivity of Salmonella typhimurium was3.0×10~2 CFU/m L,the sensitivity of S.aureus was 1.1×10~2 CFU/m L,and the sensitivity of Escherichia coli O157:H7 was 1.75×10~3 CFU/m L.3.Establishment of IMS-LAMP detection method for S.aureus,Salmonella and E.coli O157:H7.The IMS-LAMP method detects different concentrations of pure culture bacteria liquid.The minimum detection concentration for Salmonella typhimurium was 3.0×10~2 CFU/m L,and the minimum detection concentration for S.aureus was 1.1×10~2 CFU/m L.The sensitivity to E.coli O157:H7 was 1.75×10~2CFU/m L.The IMS-LAMP method and LAMP method were used to detect artificially contaminated beef samples.The sensitivity of the IMS-LAMP method for Salmonella typhimurium and E.coli O157:H7 was 1.2×10~3 CFU/m L and 7.0×10~3 CFU/m L,which were 10 times higher than the LAMP method.The sensitivity of the IMS-LAMP method of S.aureus was the same as that of the LAMP method,both of which are 4.4×10~4CFU/m L,but the positive rate of the IMS-LAMP method for detecting samples with a concentration of 4.4×10~3 CFU/m L was 66.67%.Not detected at the concentration.For low-concentration samples at 10~0CFU/m L for bacterial culture,the IMS-LAMP method can detect the amplification curve at least 2 h earlier than the LAMP method.