| Bacillus cereus (Be) is a widely spread baeterial pathogen that can infect animinals and human beings. Food such as milk, rice can easily be contaminated by Be after being exposed to air more than24h. Recently, there are a lot of studies on prevalence of contaminations by Be in raw milk, dairy, starch food, and ready-to-eat food in the world. But Be contaminations in vegetables, raw meat, edible fungi, aquatic food have little been reported. In this study, we evaluated the prevalence of contamination by Be in different foods in China The food products including raw meat, frozen food, aquatic food, ready-to-eat food, vegetables, edible fungi and dairy were collected in Xi’an, Yangling and Shenzhen, Shaoguan, Zhanjiang, Shantou, Heyuan, Haikou, Sanya, Beihai, Nanning, Fuzhou, Xiamen, Shanghai, Hefei, Nanchang, Wuhan, Chengdu, Kunming, Lanzhou, Harbin, Xi’an, Taiyuan, Beijing and Jinana. Additiona-lly, the virulence-related genes of Be and Bt strains isolated from Guangdong area were dete-cted through PCR. Finally, The Be isolates were genotyped by enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), and then the distribu-tion and genetic diversity were studied through cluster analysis. It can provide the basis for the prevention and control of food safety. Major findings and conclusions were summarized in the following:1. A total of1150samples were collected from markets and examined by qualitative and quantitative methods and269samples were positive for Be, The average positive rate was23.4%.Be contaminations of seven kinds of food retailed in supermarket and freemarket in23cities were common. The comtamination rates ranged from17.0%in aquatic food to40.4%in vegetables. The pathogen was also identified in dairy products (17.1%), edible fungi (21.7%), raw meat (22.6%), frozen food (23.4%) and ready-to-eat food (26.1%). The pollution condition for Be in different cities were also different. The contamination rates were:Hefei (48.0%) Jinan (40.0%) Lanzhou (38.0%) Beihai (36.0%)=Xiamen (36.0%) Beijing (34.0%) Taiyuan (30.0%) Shantou (26.0%)=Harbin (26.0%) Wuhan (24.0%)=Shenzhen (24.0%) Shaoguan (22.0%)=Fuzhou (22.0%)=Xi’an (22.0%) Sanya (20.0%)=Chengdu (20.0%) Heyuan(16.0%) Haikou (12.0%) Nanning(12.0%) Shanghai (10.0%) Zhanjiang (8.0%) Nanchang (6.0%)=Kunming (6.0%). Comparing Bc pollution situation of the samples from different market, the positive rate from supermarket (23.7%) was slightly higher than from freemarket (23.2%). The contamination levels of Bc in food were not high. The MPN values of43.5%of positive samples were in3to100MPN/g,7.8%was, in100to1000MPN/g, and only9.7%was more than1000MPN/g.2. Bc and Bacillus thuringiensis (Bt) are two different species in Bacillus cereus group. They have high degree of ho mo logy. To further explore the virulence of Bc and Bt to understand their potential the pathogenicity, this study used PCR to detect the Bc and Bt strains which isolated from Guangdong for the presence of nine virulence-related genes. The genes included toxin regulatory gene plcR, three hemolysin BL (HBL) genes (hblC,hblD, hblA), three nonhemolytic enterotoxin (NHE) genes(nheA, nheB, nheC), enterotoxin T gene (becT) and the emetic toxin gene (ces). The results were as follows:The detection rates of plcR, hblC, hblD, hblA, nheA,nheB, nheC, becT, ces genes in Bc were100.0%,59.6%,95.7%,100.0%,100.0%,100.0%,100.0%,85.1%,2.1%, and in Bt were98.4%,60.7%,98.4%,100.0%,100.0%,98.4%,100.0%.88.7%,0.0%.3. ERIC-PCR is a relatively simple, highly reliable, and cost-effective method, and was demonstrated to generate clear DNA fingerprints within a single bacterial species. The aim of this study was to establish ERIC-PCR amplification system which was suitable for the molecular typing of Bacillus cereus (CMCC63303). The research discussed main factors to ERIC-PCR amplification system, including dNTPs, Taq DNA polymerase, primer, DNA template, and annealing temperature through ladder experiments. On this basis, the ERIC-PCR amplification system for Bc in four levels of five factors (DNA template, Mg2+dNTP, Taq DNA polymerase, primer) were investigated by using orthogonal design and variance analysis. The optimal reaction system of ERIC-PCR was:the25μL PCR mixture including75ng of template genomic DNA,5.00mmol/L Mg2+,04mmol/L each dNTP,0.2μmol/L of each primer,0.75unit Taq DNA polymerase, and the annealing temperature of45℃.4. To further study the genetic diversity of the Bc,50Bc strains isolated from the Guangdong region were genotyped by the optimized reaction conditions of ERIC-PCR, Through cluster analysis, we found that50isolates have been divided into47types, resolution DI was0.996, similarity coefficient was between0.50-1.00. It showed a high genetic diversity and good typing results. |
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