The Research On Detection Of Food-Borne Pathogenic Bacteria By Nucleic Acid Dipstick Assay Based On LCR And PCR

Enterobacter sakazakii, Staphylococcus aureus, Escherichia coli and Salmonella are four common foodborne pathogenic bacteria. They are not fatal for healthy people, but they may cause a devastating foodborne illness. Now food safety has become a hot topic in today's society. Some detection methods such as traditional foodborne pathogenic immunology, molecular biology and so on have not met the needs of today's food safety. Therefore developing a fast, sensitive and specificity detection method has become an important trend of solving the problem of food safety.In the 1980 s, Immunochromatographic strip was gradually developed and widely favored because of its rapid detection, low cost, portable advantage and so on. However the preparation of traditional immunochromatographic strip usually needs to screen specific antibody of bacteria to be detected. The process is time-consuming and difficult, and the homogeneity among organisms of food sample can easily lead to low specificity of immunoassay. In addition, low sensitivity is another disadvantage of traditional immunochromatographic strip because there are no signal amplification in the experiment.The traditional immunochromatographic strip was improved to be the nucleic acid strip in this experiment, which was used to detect the nucleic acid of food-borne bacteria. The detection sensitivity can be improved significantly by amplification of LCR and PCR. Biotin, FAM and digoxin labeled in the nucleic acid can combine with streptavidin, anti-FAM antibody and anti-digoxin antibody in the nucleic acid strip to achieve the purpose of detection. Thus the problem of low specificity caused by screening antibody voluntarily can be avoided.The experiment mainly includes the following four parts: 1) The experimental conditions of streptavidin(SA) conjugated colloidal gold was optimized, and two kinds of nuclear acid strips( NO. 1 strip and NO. 2 strip)was prepared. The results showed that optimal pH of streptavidin(SA) conjugated colloidal gold was 7.0, and optimal concentration of SA was 0.6 mg/ml. 2) The DNA of Staphylococcus aureus was amplified by Ligase Chain Reaction(LCR), and the experimental conditions of LCR were optimized.The LCR product was detected by using NO. 1 strip, and the specificity and sensitivity of the detection method were analyzed. The results showed that the optimal concentration of probes was 0.1?M and the optimal dosage of enzyme was 2U for 15?l of LCR system. The specificity was good when other 8 bacteria except Staphylococcus aureus was detected by NO. 1 strip. The detection limit was 10 fM. 3) PCR product of Enterobacter sakazakii and two kinds of specific probes were used in NO. 1 strip after hybridization.The concentrations of probes were optimized and the specificity and sensitivity of the detection method were analyzed. The results showed that optimal concentration of probes of Enterobacter sakazakii was 0.5?M. The specificity was good when other 7 bacteria except Enterobacter sakazakii was detected by NO. 1 strip. The detection limit was 7.86 cfu/ml, and the mixture of 5.5 ×107 cfu/ml salmonella had no effect on detection sensitivity. 4) PCR product of Escherichia coli and Salmonella and four kinds of probes were used in NO. 2 strip after hybridization.The concentrations of four probes were optimized and the specificity and sensitivity of the detection method were analyzed. The results showed that optimal concentration of probes of Escherichia coli and Salmonella was 0.5?M. The specificity was good, and the detection limit was 10 cfu/ml. The mixture of 8.6×106 cfu/mL of Staphylococcus aureus had no effect on detection sensitivity.