| Listeria Monocytogenes is recognized as an important human and animal pathogen. The endangerment of Listeriosis has aroused great attention of the Department of Food and the Department of Health in the world at recent year. How to find a kind of fast, sensitive, specific, reasonable inspection method is an important research subject that the Department of food and the Department of Health to solve. In this paper, we established Polymerse Chain Reaction protocol to detect Listeria Monocytogenes. The assay was composed of two parts. The part 1 was designed to isolate and identify Listeria Monocytogenes from 165 chicken samples. The isolation and culture process adopted GB4789-93 protocol, we identified the suspicious strains by API method and then validated the result by ELISA method. The isolation results indicated that: among the 165 chicken samples, there were 16 samples have Listeria Monocytogenes and the positive rate is 9.7%. The rate of the cooked chicken was the highest (13.3%) and the rate of the peeled brisket was the lowest (8.0%). The results of ELISA method indicated that the rates of the two methods were identical. At the second assay, we established PCR protocol to detect Listeria monocytogenes. The major virulent factors of Listeria monocytogenes are Hemolytic Factor gene (hly gene), inlA gene, ActA gene etc. The most important virulent gene is hly gene.It not only can make the Listeria monocytogenes avoid devourment, and it can impair erythrocyte and hemaloblast. As this reason, we compared the hly gene sequence that have been published on GENBANK. Then we adopted the primer sequence that Fitter designed and amplified partial hly gene. Its length was about 730 bp. We have set up the PCR protocol and identified the fragment by electrophorese. The result indicated that it was the specific nucleic acid fragment. At the same time, we optimized the reaction condition, such as MgCl2 concentration, Taq enzyme quantity, dNTP concentration, primer quantity and circulating parameter. The result showed that the best reaction system of 25ul was: the best concentration of MgCl2 is 2.5 mmol/l, the best enzyme quantity is 1.25U, the best dNTP concentration is 200 umol/1, the best annealing temperature is 60.0C, the best circulating parameter is 30 cycles. This assay adopted alkaline analysis to extract DNA. The operation of this method is simple and convenient, and it is economic and fast. At the same time, we have carried out specificity, sensitivity, interference and stability test of PCR protocol. The test result showed that the detection sensitivity of this method is 7.3 cfu/ul, the detection limit is 0.038 cfu/ul and it had perfect sensitivity. We applied 8 strains ofnon-Listeria genus (such as Staphylococcus aureus, S.enteritidis, Bacillus cereus, E.coli etc) and 2 strains of L.innocna, 1 strains of L.ivanovii and 1 strain of L.welshimeri to test the specificity. The results showed that all positive bacteria have specificly amplified the purpose fragment, but other bacterium submitted negative reactions. We also applied the S.enteritidis and E.coli to test the interference. The result showed that the heterogeneous bacteria could not disturb it. We also extracted DNA and amplified the hly gene for four times at different time and all the results were positive. It indicated that the stability of this detection method was very well. We inspected 16 positive chicken samples and 35 positive strains isolated from laboratory by the PCR protocol. The results showed that all the positive samples and strains were positive. Compared the defference between assay 1 and assay 2, the PCR protocol took less time than assay 1. The entire detection course could be completed in 3 days and it is more fit for the fast inspection of food.This research indicated that we can specificly inspect Listeria monocytogenes through amplifying the partial fragment of hly gene of and it have no cross reaction with other Listeria species. This method had perfect specificity, sensitivity and stability compared with other routine met... |
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