Development Of A Colloidal Gold Strip For Rapid Detaction Of Cronobacter Sakazakii

The bacterium Cronobacter sakazakii is a food-borne pathogen which distributed in foods and the surrounding environment widely. Currently, A large number of case reports have shown that infant formula is suppose to be the major route of transmission. In recent years, due to quality problems of infant formula milk powder, the bacterium Cronobacter sakazakii in milk quality testing as level A objective pathogens received widespread attention. According to statistics, high-risk infection group of Cronobacter sakazakii is focus on newborns and immunocompromised adult, the peole who is infected may fall in severe neonatal meningitis, bacteremia and enterocolitis, death rates range from 40% to 80%. At present, the bacterium Cronobacter sakazakii detection is depend on traditional biochemical way mostly, molecular detection methods as a secondary. However, due to the biochemical detection methods are vulnerable to environmental and human’s factor that lead to interfere the results of experimental identification; molecular detection methods requires professional technicians operate the experiment, higher requirements for laboratory instruments also fail to field testing. Therefore, development of an anti-bacterium Cronobacter sakazakii colloidal gold test strip used to the field inspection is necessary. LPS are the part of cell membrane structure and function of important factor outside Gram-negative bacteria, When gram negative bacteria invade the body, LPS as a major target factor recognized by the immune system, and plays a vital role in process from infection of host cells to evade immune attack all aspects in bacteria. It has been considered as the bacterium Cronobacter sakazakii most likely virulence factors. Therefore, in-depth study on structure and receptor for LPS and seek out the pathogenic mechanism of the bacterium Cronobacter sakazakii is very necessary.The research study includes:The preparation of bacterium Cronobacter sakazakii Polyclonal Antibody:Using a standard strain ATCC51329 as immune Antigen inactivated after the high temperature, prepara anti-bacterium Cronobacter sakazakii Polyclonal Antibody by Immune 2 New Zealand White Rabbit, Antibodies through the alpha-lipoic acid-ammonium sulfate purification, potency is 1:25600 and 1:12800 respectively; the protein content of purified antibody is 2.7 mg/mL and 2.9mg/mL, respectively. Using SDS-PAGE gel electrophoresis shows good purity, mixed with seldom impurity, molecular weight of about 194kD.Extraction and purification of the bacterium Cronobacter sakazakii LPS:Select the bacterium Cronobacter sakazakii as experimental material, with hot phenol method and ultrasonic crushing method extract LPS. After the purification of LPS, Protein level was measured by Coomassie brilliant blue method and nucleic acid was measured by ultraviolet Spectrophotometric Determination.Get protein content of LPS below 0.5%; nucleic acid contents is about 1.47%, LPS yield are 1.83% and 0.654%, respectively.Preparation of colloidal gold and gold-labelled antibodies:In this study we optimized preparation conditions in preparation colloidal gold solution by controlling the pH, speed, temperature and heating time on four factors, the best preparation conditions of pH 7.5, medium-speed, temperature 95℃ and heating 1 minute after boiling, coupling conditions of work are:optimal protein 0.018mg/mL; the optimum pH for 10; buffer is pH 9.8 boric acid buffer; antibody dilution formula for 2% sucrose Borate buffer solution containing 1%BSA. Under these conditions gold labelled antibody could stable save.Assembling and evaluation of Immunochromatographic test strip:Use of the bacterium Cronobacter sakazakii Polyclonal Antibody, in colloidal gold strip preparation of Immunochromatographic test strip for testing. PVC floor, NC membrane, glassfiber membrane, absorbent pad and the sample pad Immunochromatographic test strip for assembled. Tests are immuno-detection limit to 105 cfu/mL, LPS of the detection limit for 10-4mg/mL, strip test has excellent specificity, accelerated aging in 37℃ incubator using normal after 60 d, indicate at room temperature can save at least 8 months.