Cloning, Expression Of Listeria Monocytogenes Iap Gene And Rapid Detection Of Pathogenic Listeria Spp.

Listeria is an intracellular gram-positive bacteria with very wide distribution, and can be found in many kinds of food. The pathogen can cause the infections of blood and brain tissue. Study its virulence factor is of great significance for understanding the pathogenic mechanisms and rapid diagnose. Coded by iap gene, P60 is a extracellular protein produced by Listeria spp. It is essential to cell division and closely related to the invasion of Listeria. Among the seven species, only Listeria monocytogenes and Listeria ivanovii are pathogenic. Listeria monocytogenes is a bacterial foodborne pathogen responsible for human and animal listeriosis. Listeria ivanovii can infect ruminant animal and cause losses in livestock production. The objective of this study is to (i) cloning and expression of iap gene in E.coli expression system; (ii) establish a PCR technique used for the efficient,rapid and reliable detection of L.monocytogenes and distinguishing L.monocytogenes and L.ivanovii; (iii) study on the prevalence of pathogenic Listeria spp. in different kinds of food products and sewages at Yangzhou markets.1. Cloning, expression of Listeria iap gene in E.coliThe iap gene was amplified from L.monocytogenes strain Lm03061809 by PCR. The PCR product was inserted into the high-expression vector pET 30a(+) and pGEX 6p-1 for sequencing and expressed in E.coli BL21(DE3) and BL21 respectively. It was shown that the sequence has 99.5% homogeneity to that in GenBank. The expressed protein was purified by His.Bind Resin Kit and GST purification Modules kit.The results of SDS-PAGE verified that the two desired soluble recombinant protein was expressed and the concentration of purified protein was 2.6mg/ml and 1.8mg/ml respectively.The soluble recombinant protein P60 with two different labels provided the useful material for further study.2. Establishment of rapid detection method for pathogenic Listeria spp.based on PCRPathogenic and nonpathogenic Listeria spp are coexistent and their targets are not identical. It is essential to establish a efficient and rapid detection method for L.monocytogenes and distinct between L.monocytogenes and L.ivanovii. Based on the iap gene especial for Listeria, hly gene especial for hemolytic Listeria spp. and InlB gene especial for L.monocytogenes, the PCR detection system was established. It's limit of L.ivanovii DNA template is 3.46pg and L.monocytogenes DNA template is 3.93 pg. The assay could detect as few as 10-6 cfu of pathogenic Listeria spp. in 10g of pork and 10 ml milk or water following 16h of enrichment in Listeria Enrichment broth(LEB) at 37℃.3. Prevalence of pathogenic Listeria spp.in different kinds of food products and environmentsIn this study we attempted to investigate the prevalence of pathogenic Listeria spp.in different kinds of food products and market sewages at Yangzhou markets. 630 of food product samples, 190 of sewages samples from three markets (marketA,B,C) were collected for the isolation and identification of pathogenic Listeria spp. The results showed that the overall prevalence of L.monocytogenes for food products and sewages samples in the three markets was 1.34%, with prevalence ranging from 0.00% which was found in sewages and seafood in market A and vegetables in market B, C to 3.75% which was found in fresh meat products in market B. Statistic analysis showed that the average prevalence of fresh meat (2.92%) was significantly higher than that of other products. Among the 820 samples, there was one positive sample with L.ivanovii found in mussel of market B and one positive sample in mussel of market C. The overall prevalence of L.ivanovii for food products and sewages samples in the three markets was 0.24%, with the highest prevalence of 1.43% which was found in seafood in market B. The prevalence of other types of samples was 0.00%.In addition, there were two positive samples with both L.monocytogenes and L.ivanovii. From the above results, it can be concluded that (1) there were different levels of contamination with pathogenic Listeria spp. in different kinds of food products and market sewages at Yangzhou; (2) fresh meat was more suitable for the growth and spread of L.monocytogenes than other types of foods; (3) L.ivanovii can exist in seafood. Conclusively our data will provide scientific basis for the food safety measures.