| ObjectiveThe Listeria monocytogenes are widely distributed in nature and can be found on decaying vegetable and in soils, animal feces, sewage, silage, and water. Of the lisiterial species, Listeria monocytogenes is the pathogen of concern for humans. Croups at high risk are immunocompromised people, pregnant women, newborns and elders. The clinical manifestations of listeriosis include meningitis/meningoencephalitis, septicemia, mild febrile gastroenteritis and spontaneous abortion. The mortality rate is approximately 30%. The bacteria can grow over the temperature range of about 1 C to 451 and at a pH of 4.1 to 9. 6. In general, the organism has been found in raw milk, soft cheeses, poultry, seafood, fresh and frozen meat, fruits, and vegetable products.Since 1980' s, a number of outbreaks of human listeriosis as well as sporadic cases have been reported in Canada, the United State American, Italian, et al, and a lot of people died from listeriosis every year. In China, some kinds of food were contaminated by Listeria monocytogenes.Many international organizations have been interesting in Listeria monocytogenes , which is one of four food - borne bacterium. Some countries have established legal limits on the number of Listeria monocytogenes that are permissible in food. AOAC, FDA and China are using conventional detection methods for Listeria monocytogenes include selective enrichment, isolation of colonies on agar and confirmation using hemolysin activity and other biochemical reaction as diagnostic tests. These methods are laborious and it takes 4-7 days to achieve a confirmed identification, which can not adapt for the need of rapid, specific and sensitive detection and disadvantage for food administration and disease prevention. Thus, it is urgent to establish a rapid, specific and sensitive detectionmethod for Listeria monocytogenes.In present, polymerase chain reaction ( PCR) is a detection method used extensively. We are going to choose the listeriolysin 0 gene as the target for Listeria monocytogenes - specific primers, and optimize detective condition of PCR for this bacteria and establish a detective method of LM by PCR in food.Methods1. Establishment of PCR method1.1 DNA extraction: Promega DNA purification kit.1.2 Optimization of PCR amplificated condition: the appropriate magnesium concentration and annealing temperature.1.3 Gel electrophoresis of PCR productsThe PCR products were analysed by gel electrophoresis using 1.2% agarose gels in 0.5 x TBE buffer. Gels were run for 60 min at 100V in TBE buffer containing 0. 5g/ml ethidium bromide to enable visualization of PCR products by u. v. transillumination. Molecular weight markers were included on each gel (100bp DNA ladders, Promega).1.4 Measuring sequence of DNA fragment.2. Preparing for bacterium2.1 Listeria monocytogenes from national institute for the control of pharmaceutical and biological products were grown in Listeria enrichment broth overnight in 311.2.2 Other species were grown in nutrient broth overnight in 37t.3. Sensitivity of detection of Listeria monocytogenesSerial decimal dilutions were prepared from the initial Listeria enrichment broth in that cells were cultured in for 24h. DNA should be extracted from different dilution, and be prepared for amplification.4. Specificity of amplification of primers by PCRThere were 105 bacterial strains to be detected including 62 Listeria monocytogenes, 3 Listeria innocua and 40 other bacterial strains except for Listeria strains by PCR.5. Artificially contamination in food by Listeria monocytogenes5.1 Preparing for samples25 g of milk, raw meat, frozen shrimp and cabbage were transferred into the homogeneous bag with 225ml Listeria enrichment broth, respectively.5.2 Method of artificially contaminationMilk, raw meat, frozen shrimp and cabbage were artificially contaminated with an overnight culture of by adding 10 - fold dilutions. Aliquots were then subjected to DNA isolation. The bacterial number in... |
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