Construction And Application Immunomagnetic Separation And Loop-mediated Isothermal Amplification(IMS-LAMP) For The Detection Of Three Foodborne Pathogens

Food may be contaminated by foodborne pathogens in many links such as production,processing,transportation,sales and cooking.Foodborne pathogens have become an important source of food safety problems.Vibrio parahaemolyticus,non-O1/O139 Vibrio cholerae and Vibrio fluvialis are common foodborne pathogens.If eaten by mistake,it may cause diarrhea,abdominal pain and even life-threatening in serious cases.At present,most of its detection methods have the problems of long detection time,complex and expensive instruments and cumbersome operation.Therefore,it is urgent to establish a convenient and fast method to detect these foodborne pathogens.Immunomagnetic separation（IMS）technology combines the unique advantages of magnetic beads with the high specificity of immunological reaction.It can directly separate and enrich the target bacteria in complex matrix or enrichment solution,effectively eliminate matrix interference and improve detection efficiency.It is a simple and convenient sample pretreatment method.Loop-mediated isothermal amplification（LAMP）technology has the characteristics of strong specificity,high amplification efficiency,short reaction time,simple detection method and unwanted complex equipment.It is very suitable for the rapid detection of foodborne pathogens.In this study,the IMS-LAMP detection method of foodborne pathogens was established by combining immunomagnetic bead separation technology with Loop mediated isothermal amplification method.It is simple,rapid,sensitive and convenient for field detection.The specific experimental results are as follows:（1）An immunomagnetic separation method for V.parahaemolyticus,non-O1/O139 V.cholerae and V.fluvialis was established.Immunomagnetic beads were synthesized by polyethyleneimine glutaraldehyde mediated method.When preparing V.parahaemolyticus immunomagnetic beads,non-O1/O139 V.cholerae immunomagnetic beads and V.fluvialis immunomagnetic beads,the optimal antibody addition of 1 mg 250 nm carboxylated magnetic beads was 125,100,100μg respectively.At the same time,the enrichment conditions of V.parahaemolyticus immunomagnetic beads,non-O1/O139 V.cholerae immunomagnetic beads and V.fluvialis immunomagnetic beads were optimized.The optimal addition amounts of immunomagnetic beads were 0.6,0.6 and 0.8 mg respectively,and the optimal capture time was 45 min.When the concentration of miscellaneous bacteria is about10 times higher than the target bacteria,its capture efficiency is almost unchanged,it shows that the prepared immunomagnetic beads have strong ability to resist the interference of miscellaneous bacteria.At the same time,when the concentration of bacterial solution is as low as 10¹ CFU/m L,it can still be captured and has good sensitivity.（2）Establishment of LAMP for V.parahaemolyticus,non-O1/O139 V.cholerae and V.fluvialis.The tox R protein sequences of Vibrio were selected through NCBI.After the protein sequences of variable region were obtained through multi sequence alignment,specific amplification primers were designed and screened.Then the LAMP reaction system was established by optimizing the Mg²⁺concentration,enzyme concentration,reaction temperature and reaction time in the reaction process,The specificity and sensitivity of each primer were detected.The results showed that in the V.parahaemolyticus LAMP system,the optimum concentration of Mg²⁺was 6m M and the optimum concentration of enzyme was 0.32 U/μL,the optimum reaction temperature was 63.5℃and the amplification time was 30 min;In non-O1/O139 V.cholerae LAMP system,the optimum concentration of Mg²⁺is 4m M and the optimum concentration of enzyme is 0.32 U/μL,the optimum reaction temperature was 60.3℃and the amplification time was 30 min;In the V.fluvialis LAMP system,the optimum concentration of Mg²⁺was 4 m M and the optimum concentration of enzyme was 0.24 U/μL,the optimum reaction temperature was 63℃and the amplification time was 40 min.The sensitivity of V.parahaemolyticus,V.cholerae and V.fluvialis were 1.9×10⁰,6.2×10¹ and 7×10⁰ CFU/m L.（3）Establishment of IMS-LAMP method for the detection of V.parahaemolytic-us,non-O1/O139 V.cholerae and V.fluvialis.The previously established immunomagnetic bead enrichment method and Loop-mediated isothermal amplification detection method were combined,and the fluorescent dye SYTO-9was used to realize the visual detection of the detection results.The detection sensitivity and specificity of water samples and fish samples were studied.The experimental results show that the established IMS-LAMP detection method has good specificity.The results of agarose gel electrophoresis showed that the detection sensitivity of V.parahaemolyticus,non-O1/O139 V.cholerae and V.fluvialis in water samples was 1.2×10¹,2.5×10¹ and 4.0×10⁰ CFU/m L respectively,and the detection sensitivity in fish samples was 3.2×10³,2.5×10²,4.1×10¹ CFU/m L,respectively.After using fluorescent dye SYTO-9,the detection sensitivity in water samples of V.parahaemolyticus,non-O1/O139 V.cholerae and V.fluvialis was1.2×10⁰,2.5×10⁰,4.1×10⁰ CFU/m L respectively.The detection sensitivity of fish samples was 3.2×10¹,2.5×10¹ and 4.1×10⁰ CFU/m L,respectively.The use of fluorescent dyes increases the detection sensitivity by 10-100 times.