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Yang Y.,Nanchang University | Xu F.,Sino German Joint Research Institute | Xu H.,Nanchang University | Aguilar Z.P.,Ocean NanoTech, LLC | And 8 more authors.
Food Microbiology | Year: 2013

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 102 CFU/ml (1.2 × 102 CFU/ml for S. Typhimurium, 4.0 × 102 CFU/ml for E. coli O157:H7 and 5.4 × 102 CFU/ml for L. monocytogenes) in pure culture and 103 CFU/g (5.1 × 103 CFU/g for S. Typhimurium, 7.5 × 103 CFU/g for E. coli O157:H7 and 8.4 × 103 CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use. © 2013 Elsevier Ltd.


Yang Y.,Nanchang University | Wan C.,Sino German Joint Research Institute | Xu H.,Nanchang University | Lai W.,Nanchang University | And 7 more authors.
Food Control | Year: 2012

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10 6 CFU/ml in pure culture or 1 × 10 6 CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10 2 CFU/ml (4.3 × 10 2 CFU/ml for S. Typhimurium, 3.7 × 10 2 CFU/ml for S. Paratyphi B, 7.2 × 10 2 CFU/ml for S. Typhi) in pure culture and 10 3 CFU/g (4.3 × 10 3 CFU/g for S. Typhimurium, 3.7 × 10 3 CFU/g for S. Paratyphi B, 7.2 × 10 3 CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce. © 2012 Elsevier Ltd.


Yang Y.,Nanchang University | Wan C.,Sino German Joint Research Institute | Xu H.,Nanchang University | Aguilar Z.P.,Covance Laboratories Inc. | And 5 more authors.
Microbes and Infection | Year: 2013

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (. S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine. © 2013 Institut Pasteur.


Yang Y.,Nanchang University | Wan C.,Sino German Joint Research Institute | Xu H.,Nanchang University | Wei H.,Nanchang University
Vaccine | Year: 2013

Salmonella is gram-negative flagellated bacteria that can cause food and waterborne gastroenteritis and typhoid fever in humans. Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined, the effectiveness of the currently available vaccines is also limited. Outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against salmonellosis. OMPs interface the cell with the environment, thus representing important potential vaccine candidate for pathogen infection. We showed that the outer membrane porin L (OmpL) is a transmembrane β barrel (TMBB) protein, which forms 12 transmembrane β-strands. OmpL of S. Typhimurium is highly immunogenic, OmpL could evoke humoral and cell-mediated immune responses, and confer 100% protection to immunized mice against challenge with very high doses of S. Typhimurium. Besides, very efficient clearance of bacteria from the reticuloendothelial systems of immunized mice was seen. The homology search further revealed that OmpL is widely distributed and conserved, homologous proteins were identified in S. Typhi and Paratyphi by RT-PCR and western blot. We also found that anti-rOmpL serum harber a high bactericidal activity for Salmonella serovars tested in this study. Therefore, OmpL provide a promising target for the development of a candidate vaccine against Salmonella infection. © 2013 Elsevier Ltd.

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