Establishment Of Immunomagnetic Bead Detection Method For Salmonella Enteritidis

Salmonella enteritidis,an important zoonotic pathogen,widely spreads in the horizontal and vertical farms.It not only causes huge economic losses to farms,but also poses a threat to food safety due to pollution of livestock and poultry products.Therefore,prevention and control of this bacteria is of great important to public health significance.The key to preventing and controlling Salmonella enteritidis lies in its rapid diagnosis.Currently,the main methods for detecting Salmonella include the national standard GB 4789.4-2016detection method,conventional PCR,and high performance liquid chromatography.However,there are many disadvantages such as long detection cycle,cumbersome steps,low efficiency and high investment,which cannot meet the detection requirements of the breeding industry and livestock and poultry products.Thus,a simple and economical method with high sensitivity and good specificity is needed to be urgently established,in order to prevent and control Salmonella enteritidis more practical.To make up the above-mentioned detection method for Salmonella enteritidis,216clinical samples of duck,fowl and swines origin from Shanghai,Anhui,Shenyang and Shandong were collected and 33 strains of Salmonella enteritidis were sucessfully isolated and identified in this study.In order to expand the epitope of Salmonella enteritidis,four strains(JA070,JA133,SM012,SM013)and two standard strains(ACTT13076,CMCC50041)of Salmonella enteritidis were selected and mixed as the immunized strains.The specific monoclonal antibodies against Salmonella enteritidis were successfully obtained by immunizing Balb/C mice.A new detection method of Salmonella enteritidis based on immunomagnetic beads was established by using the prepared monoclonal antibody.The results are as follows:1.Isolation of clinical samples of SalmonellaIn order to identify the three common clinical Salmonella serotypes,the specific genes inv A(Salmonella),sdf I(Salmonella enteritidis),Stm4495(Salmonella typhimurium)and SPUL-2693(Salmonella pullorum)were selected,and the specific primers were designed.The quadruple PCR detection method of Salmonella was successfully established by optimizing the system.This method has good specificity,and the detection limit was 500pg/ml for genome and 10~3 CFU/ml for bacterial solution,showing the high sensitivity of this method.This method was used to detect 216 clinical samples of duck,fowl and swines origin from Shanghai,Anhui,Shenyang and Shandong.63 strains of Salmonella were successfully isolated and identified,including 33 strains of Salmonella enteritidis.A foundation was laid for the preparation of monoclonal antibodies against Salmonella enteritidis through this study.2.Preparation of monoclonal antibody against Salmonella enteritidisSix strains of Salmonella enteritidis(ATCC13076,CMCC50041,JA070,JA133,SM012,SM013)were selected and inactivated by formaldehyde.Balb/C mice were immunized with the same amount of mixed Salmonella enteritidis.Spleen cells of immunized mice were collected and fused with SP2/0.After the cell supernatant was obtained,monoclonal cells with high titer and good specificity were screened by indirect ELISA and then ascites was prepared.In this study,a high titer and specificity monoclonal antibody cell line of Salmonella enteritidis was screened and named as 8H.The results of ELISA showed that the titer of the monoclonal antibody against Salmonella enteritidis was more than1024000.The specificity test showed that the monoclonal antibody against Salmonella enteritidis had no cross reaction with E.coli,Pseudomonas aeruginosa,Vibrio parahaemolyticus,Listeria monocytogenes and Enterobacter sakazakii.The results of antibody typing showed that the monoclonal antibody was light chain Kappa type and heavy chain Ig G1 type.This study laid a foundation for the establishment of immunomagnetic bead detection method for Salmonella enteritidis.3.Establishment of an immunomagnetic bead detection method for Salmonella enteritisIn order to establish an immunomagnetic bead detection method for Salmonella enteritidis,the monoclonal antibody against Salmonella enteritidis was coupled with carboxyl magnetic beads,and the test conditions were optimized from the four aspects of magnetic bead diameter,coupling buffer,capture system and capture time.The results showed that the capture efficiency reached the highest when the coupling buffer mest(0.025m,p H 7.0)was selected,the diameter of magnetic beads was 180 nm,and the capture time was 45 min.The results of specific capture test showed that the capture rate of six strains of Salmonella enteritidis was as high as 95%,while the capture rate of other seven common clinical pathogenic bacteria was less than 10%,which indicated that the immunomagnetic bead detection method established in this study had good specificity.The results of sensitivity test showed that the capture rate could still reach 88%when the bacterial count was 10~2CFU/ml,which indicated that the immunomagnetic bead detection method established in this study was highly sensitive.Salmonella enteritidis(CMCC50041)was added into the liver grinding fluid to simulate clinical samples.When the bacterial counts were 10~3 CFU/ml and10~2 CFU/ml,the capture rates reached 91.55%and 87.75%respectively,which provided technical support for the rapid detection of Salmonella enteritidis in clinical samples and livestock and poultry products.In this study,we successfully prepared monoclonal antibody against Salmonella enteritidis,and established an immunomagnetic bead detection method for Salmonella enteritidis based on the antibody,which provided technical support for the detection of Salmonella enteritidis...