A Rapid Detection Method Of RPA-LF Of Staphylococcus Aureus Based On Immunomagnetic Separation

As a foodborne pathogen,Staphylococcus aureus(SP),widely exists in the environment.It is prone to contaminate meat,vegetables and other foods.Due to the lack of effective,rapid and visual detection methods,food safety incidents caused by Staphylococcus aureus occur from time to time.Therefore,it is urgent to establish a rapid and sensitive detection method which can be applied in large quantities.At present,the detection methods of Staphylococcus aureus generally have a long time and need expensive equipment,which is not conducive to mass detection at the grassroots level.Immunomagnetic separation(IMS)can significantly concentrate the targeted bacteria,reduce the interference of food substrate and others bacteria in the later detection,and improve the detection efficiency.Recombinase polymerase amplification technology(RPA)has the advantages of more rapid,convenient,efficient and cheap.This research established an RPA-LF rapid detection technology for Staphylococcus aureus based on immunomagnetic separation.It will provide a new technical solution for the detection of S.aureus.In order to prepare specific monoclonal antibodies that can be used for the detection of S.aureus,4strains of Staphylococcus aureus from different sources(ATCC6538,ATCC29213,CMCC26003 and SH001)inactivated by formaldehyde were used as the whole bacterial immunogen to immune BALB/c mice in this study.After the fusion of mice spleen cells and SP2/0 cell,indirect ELISA be used to screen monoclonal cells with high titer and good specificity.After subcloning,the cells were injected into the peritoneal cavity of BALB/c mice to collect ascites.In this study,a total of 4 monoclonal antibodies were screened with good specificity and high titers,among which the titer of SP4 antibody was higher than 1:5.12×10~6.The light chains of the four antibodies were all Kappa type,the heavy chains of SP2were IgG1 type,and the heavy chains of SP1,SP3 and SP4 were IgG2 type.The results of competitive EILSA showed that there was no epitope crossover or coverage between the four monoclonal antibodies.The monoclonal antibody prepared in this study have good characteristics,which laid a foundation for the development of Staphylococcus aureus Immunomagnetic separation in the later stage.Monoclonal antibody Conjugated with carboxyl magnetic beads can be used to prepare Immunomagnetic beads for bacteria capture.In order to optimize the conditions of immunomagnetic beads,the effects of four conjugating buffers,three different diameters of magnetic beads and the capture time were studied.The results showed that when the conjugating buffer was MEST(0.025 M,pH 7.0),the capture efficiency was the highest,the best magnetic bead diameter was 750 nm,and the capture rate reaches the highest after the capture time is over 45min.The specific capture experiment showed that the capture rate of immunomagnetic beads for S.aureus was above 90%,while that for others 12 bacteria was below 10%,indicating that the immunomagnetic beads had high capture rate and good specificity.The sensitivity detection of the immunomagnetic beads showed that the S.aureus could still be successfully captured when the bacteria in the capture system was 10~2 CFU.The immunomagnetic beads prepared in this study had good characteristics,which laid a foundation for the rapid detection of S.aureus in food substrate in the later stage.In order to establish an IMS-RPA-LF detection method for rapid detection of S.aureus in milk.In this study,RPA primers,incubation temperature and incubation time is optimized,and screened out a pair of primers that has the best detection effect against S.aureus thermostable nuclease nuc gene,its best incubation temperature is 39℃,optimal incubation time is 10 min.The specificity study showed that RPA-LF had no cross-reaction with 12 common food-borne pathogens.The results of the sensitivity test for RPA-LF indicated that the lowest detection limited of the genome of S.aureus was 600 fg.In order to evaluate the detection performance of IMS-RPA-LF on actual samples,this study conducted a simulation test by adding different amount of S.aureus in milk.The results showed that when the concentration of S.aureus was 7.5×10~2 CFU/mL in milk,IMS-RPA-LF could detect positive.The above results indicate that the RPA-LF detection method established immunomagnetic separation in this study has the advantages of high specificity,short detection time and high detection sensitivity,and this study provides a reference for the development of detection methods for S.aureus.