Construction Of Rapid Detection Method As Well As Screening And Verification Of Specific Antigen For Cronobacter

Cronobacter spp. is an important opportunistic foodborne pathogen. It could infect pre-term neonates and infants, and cause severe forms of meningitis, necrotizing enterocolitis or bacteremia with a mortality rate of 40%-80%. Impairment of mental and physical for most people, who suffered Cronobacter-associated meningitis, is permanent and irreversible. Although the transmission vehicle of Cronobacter associated with its infection has not been confirmed, evidence from many clinically diagnosed cases and epidemiological studies suggested that infant formula products could be the potential sources of Cronobacter. Therefore, some methods used for Cronobacter detection should be established, which are important and neccessary to monitor the Cronobacter contamination in food processing. The aim of this study is to develop a rapid, sensitive, accurate, simple and convenient method for detection of the Cronobacter in infant formula samples, and to screen the Cronobacter-specific proteins, which are meaningful for Cronobacter identification and detection.In this study, using nano-magnetic beads and the combination of immunology technique with molecular biology technology, the capturing and detection methods for Cronobacter were established in practical infant formula samples.Cell debris of Cronobacter was used as antigen for the rabbit immunization, and antiserum with 106 titer was prepared. Then the polyclonal antibodies (pAbs) were incubated with Vibrio parahaemolyticus and Salmonella Typhimurium cells to remove the cross-reaction of pAbs efficiently, following purification by using caprylic acid and ammonium sulphate (CA-AS) method. The pAbs were conjugated on the surface of magnetic beads for the preparation of immunomagnetic beads, which were capable of recognizing and capturing the Cronobacter cells specifically. The detection limit was 5 CFU/mL of Cronobacter cells in infant formula, when combined the immunomagnetic beads capturing protocol with nested PCR. Similarly, that limit was 103 CFU/mL of Cronobacter cells in infant formula by the combination of nuclear acid probe-magnetic beads capturing and nested PCR assay. Moreover, the detection of both combinations was not influenced by the interference of other bacteria in high density, indicating their useful potential in the detection of Cronobacter in food.Anti-Cronobacter monoclonal antibody was prepared, and labeled with colloidal gold and fluorescent microsphere respectively. Two strips, namely, the colloidal gold immunechromatographic strip and fluorescent microsphere immunochromatographic strip were evaluated with artificially contaminated infant formula samples. Results indicated that the detection limit of two strips was as low as 106 CFU/mL and 105 CFU/mL, respectively, by a reaction time in 15 min. Similarly, the detection limit was not influenced by the interference of other bacteria in infant formula samples also, providing the technique basis for the commercial development of the testing strips for Cronobacter.In order to screen and determine the specific antigen of Cronobacter, monoclonal antibody for several Cronobacter proteins (a-glucosidase, OmpA and CP783) was prepared. However, the antibody specificity results showed that these monoclonal antibody cross-reacted with some other non-Cronobacter strains, indicating that the monoclonal antibodies prepared in this study were not suitable for specific detection of Cronobacter.To improve the qualitative and quantitative detection protocol for Cronobacter, 4 protein antigens of Cronobacter i.e. ATPsyn, Maltoporin and CP783, OmpA were screened by Western Blotting, using non-cross-reaction polycolonal of Cronobacter and the whole surface proteins of Cronobacter. Thereof ATPsyn, Maltoporin and CP783 were cloned and expressed according to the routine molecular method. Protein of CP783 could be regarded as specific antigen of Cronobacter by Western Blotting and its polyclonal antibodies were prepared. Furthermore, the antigen epitope of a-gluconsidase, OmpA and P783 was fully analyzed,3 specific peptides were selected as antigens, and correspondingly,3 monoclonal antibodies were prepared. Nevertheless, all of the monoclonal antibodies recognized not only Cronobacter, but also partial non-Cronobacter, indicating non-useful for specific detection of Cronobacter.So far, the report on immunological research for Cronobacter is rare. This study is helpful for the development of rapid detection method of Cronobacter, providing some new valid protocols for monitoring the distribution of Cronobacter. This study is not only a preliminary exploration for screening and verification of specific antigen of Cronobacter, but also a solid basis for further studying the specific antigen of Cronobacter in the future.