| China is the largest pork producer and consumer with the largest pork market in the world. However, in the process of production and processing of pork,the risk of contamination of pathogens easy to lead to food safety problems. There are common pathogens in pork including Salmonella, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica and Escherichia coli 0157. The key to assure meat safety is to establish a method of rapid detection of these pathogenic bacteria.Therefore, this study was conducted to provide reliable technical method and the theory basis for rapid detection and risk assessment of pathogens of pork. Firstly, the method of a rapid detection of pathogenic bacteria in pork by multiplex PCR was established following pathogens on pork to conduct an investigation in the markets. Secondly, the study was to develop real-time PCR (SYBR Green and TaqMan) method for the detection of S. aureus in pork. Finally, modelling the growth dynamics of S. aureus on pork by real-time PCR (SYBR Green) assay. The contents and results of research are as follows:1. Rapid and simultaneous analysis of five pathogens of pork using multiplex PCR1.1 Rapid and simultaneous analysis of five pathogens in pure culture using multiplex PCR.The objective of this study was to develop a rapid multiplex PCR method which would allow the simultaneous detection of five major pathogens(Salmonella, S. aureus, L. monocytogenes, Y. enterocolitica and E. coli O157) likely to be found in pork. Five pairs of primers were designed to identify invA gene, nuc gene, hlyA gene, ail gene and rfbE gene for Salmonella, S. aureus, L. monocytogene, Y. enterocolitica and E. coli 0157, respectively. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. On the basis of determining specificity of the multiplex PCR, and the renaturation temperature and number of cycles optimized, sensitivity was performed in culture media. The results suggested that the multiplex PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. A detection limit was less than 10 cf·mL-1 for detection of Salmonella, L. monocytogene and E. coli 0157, and 10 cfu·mL-1 for detection of S. aureus and Y. enter ocolitica, and 103 cfu-mL"1 for the simultaneous detection of the five target pathogens.1.2 Rapid and simultaneous analysis of five pathogens in artificially contaminated pork using multiplex PCR. Based on the better result of the previous study of the multiplex PCR compared with the traditional incubation method, sensitivity was performed with an overnight enrichment step in pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The multiplex PCR protocol successfully detected all five organisms inoculated overnight together on pork. The detection limit by multiplex PCR after enrichment for Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli 0157 inoculated individually to pork were 59,23,3,12 and 35 cfu·(25g)-1, respectively. The detection limit by multiplex PCR after enrichment for Salmonella, S.aureus, L. monocytogenes, Y enterocolitca and E. coli 0157 inoculated together to pork were detected at levels of 142,51,9,33 and 670 cfu·(25g)-1, respectively. The assay of the detection of five pathogens of pork by multiplex PCR was strong specificity, high sensitivity and high detection efficiency compared with traditional culture method. The multiplex PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.1.3 An investigation on contamination of five pathogenic bacteria from pork market. The investigation on contamination of the main pathogenic bacteria in pork (165) from June 2012 to May 2012 in Nanjing city. Based on the previous method of multiplex PCR detected pathogens in pork, the contamination of the main pathogens of porkSalmonella, S. aureus, L. monocytogenes, Yenterocolitca and E. coli 0157) was investigated from supermarkets and farmers markets in Nanjing city. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The accuracy of the method was simultaneously validated and evaluated by comparison with traditional culture methods. Firstly, the results showed that the detection rate (27.27%) of pathogens in pork using multiplex PCR was higher than that of the traditional culture method (24.8%). Athough there was significantly different for the contamination of different pathogens in pork, the positive detection rates of S. aureus of pork was the highest (12.7%). Secondly, Based on the traditional culture method, the accuracy was 82%; Based on the multiplex PCR, the accuracy was 90%. the positive detection rate (40.0%) of foodborne pathogens in pork from Farmers’market was significantly higher than that of the detecting rate (10.9%) from the supermarket. Finaly, as the survey time of the pathogens in pork was different, that of the contamination rate was significant differences. The detection rate (more than 60%) of pathogens in pork in July and August was significantly higher than that of the detecting rate in other months. However, the detection rate (less than 6.7%) of pathogens in pork from January to March and from October to December was the lowest for the whole year, multiplex PCR detection assay was strong specificity, high sensitivity and high detection efficiency compared with traditional training method.2. Rapid and reliable detection of S. aureus of pork using real-time PCR2.1 Rapid and reliable detection of S. aureus in pure culture using real-time PCR. S. aureus in pure bacterial culture was detected using real-time PCR methods without the need for pre-enrichment steps. On the basis of determining specificity and sensitivity (DNA and a cell) of real-time PCR, the standard curve (DNA and a cell) contracted was performed. Firstly, the results suggested that real-time PCR was an effective procedure having high specificity for the detection of nuc gene of S. aureus in pure culture. Secondly, although the specificity of real-time PCR (TaqMan) is better than real-time PCR (SYBR Green), the sensitivity of real-time PCR (SYBR Green) is slightly higher than real-time PCR (TaqMan). The sensitivity were 10 cfu·mL-1 for real-time PCR (SYBR Green) and 102 cfu·mL-1 for real-time PCR (TaqMan). The sensitivity of pure bacteria cultures was consistent with that of bacterial genome DNA. Based on the standard curve constructed by real-time PCR, the linear correlation and repeatability are good, and the coefficient of variation is very small. As a whole, compared with real-time PCR (TaqMan), Ct value of real-time PCR (SYBR Green) was small, so it had high sensitivity. The standard curve of pure bacteria cultures was consistent with that of bacterial genome DNA.2.2 Rapid and reliable detection of S. aureus in artificially contaminated pork using real-time PCR. The objective of this study was to develop a real-time PCR method for the detection of S. aureus in pork, while the accuracy of the method was evaluated by comparison with traditional culture methods in artificially contaminated pork. Based on DNA standard curve of pure bacteria cultures, S. aureus in pork was detected using real-time PCR methods without the need for pre-enrichment steps. Owing to the high specificity, the wide dynamic range of detection and the high sensitivity proved in pork, the real-time PCR (SYBR Green) is a useful diagnostic tool for routine quantitative detection of S. aureus of pork for improving pork safety.3. Modelling the growth dynamics of S. aureus of pork by real-time PCROn the basis of previous results of detection of S. aureus in pork using real-time PCR, we inoculated pork with S. aureus and developed primary growth models (modified Gompertz, Logistic and Richards model) of in pork at constant temperatures (7,10,15,20, 25 and 30℃) using DNA-based real-time PCR. Based on this basis, we constructed secondary model using Rathowsky (square-root) model. Compared with primary model and secondary model acquired by traditional microbiogical method, the models using real-time PCR were able to provide theoretical support to look upon real-time PCR as a tool for forecasting model. Results demonstrated that three primary growth models could be able to identify the growth of S. aureus in pork, which were certified by correlation coefficient (R2), the mean square error (MSE), bias factor (Bf) and accuracy factor (Af) values. It was manifested that the models were all effective and reliable, where modified Gompertz model was looked upon as the best one to identify the growth of S. aureus in pork. The secondary model founded on Rathowsky (square-root) model could considerably identify growth influenced by temperature. The growth rate made greater following temperatures increased and changed from 0.04 to 0.9 (log cfu·(mL·h)-1), and the lag time made less from 33 to 1 h. By comparison to the curve charactistics and growth parameters, there was no difference between models acquired by real-time PCR and traditional microbiological method. These two methods could forecast the growth of S. aureus in pork accurately. Forecasting model acquired by real-time PCR overcomed the time-consuming and laber-intensive problem, and this approach could be able to provide the theory basis for the forecasting growth and risk assessment of S. aureus in pork.In conclusion:(1) A rapid multiplex PCR method of pork that would allow the simultaneous detection of five major pathogens likely to be found in pork (Salmonella, S. aureus, L. monocytogenes, Y. enterocolitica and E. coli O157). The detection limit by multiplex PCR after enrichment for Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli 0157 inoculated together to pork were detected at levels of 142,51,9,33 and 670 cfu·(25g)-1, respectively. The assay of the detection of five pathogens of pork by multiplex PCR was strong specificity, high sensitivity and high detection efficiency compared with traditional culture method.(2) Real-time PCR was an effective procedure having high specificity for the detection of nuc gene of S. aureus in pork. although the specificity of real-time PCR (TaqMan) is better than real-time PCR (SYBR Green), the sensitivity of real-time PCR (SYBR Green) is higher than real-time PCR (TaqMan). Both the sensitivity detected and the standard curve constructed by real-time PCR of pure bacteria cultures were consistent with that of bacterial genome DNA. Real-time PCR (SYBR Green) is a useful diagnostic tool for routine quantitative detection of S. aureus of pork for ensuring pork safety.(3) Three primary growth models (modified Gompertz, Logistic and Richards models) could be able to identify the growth of S. aureus in pork. The secondary model founded on Rathowsky (square-root) model could considerably identify the influence of temperature on growth. By comparison to the curve charactistics and growth parameters, there was no difference between models acquired by real-time PCR and traditional microbiological method. These two methods could predict the growth of S. aureus in pork accurately. Forecasting model acquired by real-time PCR overcomed the time-consuming and laber-intensive problem, and this approach can be looked upon as a replacement for the conventional predictive models. |
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