| Salmonella and Escherichia coli 0157: H7 are pathogens that pose a serious threat to animals and human. However, the standard detection method is based on the traditional culturing, which has many problems such as long testing time, complicated operation and requirement of professional skills. Therefore, it is necessary to develop rapid, simple and specific detection methods. In this study, the method of helicase thermostatic amplification (HDA) combined with nucleic acid chromatography strip was established for the detection of Salmonella and E. coli 0157: H7. The method is simple and rapid, and can be used to preliminarily detect food samples that contaminated by low dose of the two pathogens.The specific gene fragments of Salmonella and E. coli 0157:H7 were selected as templates for primers designing. After optimizing amplification conditions, isothermal amplification detection methods were developed. The specific primers were designed for the amplification of invA gene of Salmonella. Optimization experiments were used to determine the optimal amplification conditions, which were finally determined as: 85 nM primers, 4 mM MgSO4, 0.7 mM dNTP, reaction at 64 ?. The specific primers were designed for amplification of rfbE gene of E. coli 0157:H7. The optimal amplification conditions were also determined using optimization experiments, which were finally determined as: 85 nM primers, 3 mM MgSO4, 0.9 mM dNTP, reaction at 67 ?.After optimization, the optimal method for strip construction was determined. Three kinds of colloidal gold particles with different diameters were prepared using trisodium citrate reduction method. The gold labled antibody was prepared by optimizing the amount of labeled antibody and pH value. And then the dilution factor of gold labled antibody, detection line coating amount, nitrocellulose membrane type, gold labeled antibody treatment solution and sample treatment solution were optimized. Based on color and sensitivity of the test strip, the optimal experimental conditions were finally determined as: 20 nm colloidal gold particles, 5 folds dilution of gold conjugated anti-digoxin antibody using pH 8.5 solution, Millipore HF 135 which was coated with 2 mg/mL of streptavidin as detection line and 80-fold diluted goat anti-rat antibody as control line, 0.5% Tween-20 as treatment solution for gold labled antibody pad, TE buffer solution as the sample developing solution.Isothermal amplification-strip detection methods for Salmonella and E. coli 0157:H7 were developed through combination of isothermal amplification with colloidal gold detection strip. Sensitivity, specificity, stability and performance for real food samples of the detection methods were further evaluated. The results indicated that the detection limits of the Salmonella detection method for DNA and pure cultured bacteria were 80.7 fg and 45 CFU/mL,respectively. Existence of other bacteria had no influence on the detection sensitivity. Specificity analysis indicated that no cross reaction was observed with Salmonella,Staphylococcus aureus,Listeria monocytogenes, Enterobacter aerogenes,Shigella and Campylobacter jejuni for the Salmonella detection method. The results of real food samples detection showed that 100 CFU/g of Salmonella contamined in chicken products and infant nutritional cereal could be detected after 2 h enrichment. Same amount of Salmonella contamined in milk could be detected after 4 h enrichment. For E. coli 0157:H7, the detection limits for DNA and pure cultured bacteria were 78.5 fg and 35 CFU/mL, respectively. Existence of other bacteria had no influence on the detection sensitivity. Specificity analysis indicated that no cross reaction was observed with Salmonella, S. aureus,L. monocytogenes,E. aerogenes, Shigella and C. jejuni. Real sample analysis indicated that 100 CFU/g of E. coli 0157:H7 contamined in chicken products and infant nutritional cereal could be detected after 2 h enrichment. Same amount of E. coli 0157:H7 contamined in milk could be detected after 4 h enrichment.The detection method developed in this study is simple, sensitivity, specific, efficient,and without requirement of instruments. The mothod developed in this study is suitable for on site rapid detections. |
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