Detection Methods In Food-borne Proteus By Polymerase Chain Reaction

Based on the increasing trend of food-borne Proteus poison, detection methods with rapidity and sensitivity should be set up for control of this bacterium. Three types of conventional polymerase chain reaction (PCR) methods and one type of newly fluorescent real-time PCR were developed to detect and identify the sampled 66 species of Proteus by comparing with the traditional plate-culture and the Vitek auto microbe system methods. Results obtained are shown as followings:Food-borne illnesses study from 264 cases reported by 218 papers published during 1962-2007 in China showed that the number of food infection via Proteus went up gradually from 1980s, 1990s to the present 21st century, with the two dominant provinces Guangdong and Shandong, two peaks of prevalence seasons the summer and the autumn, target of animal food, particularly cooked meat products, caused mainly by Proteus mirabilis and Proteus vulgaris. Detection methods used for Proteus in the reported 187 cases were traditional plate-culture method and Vitek auto microbe system which accounted for 94.65% and 5.35%, respectively. No any report of using PCR as a detection method to identify Proteus.Samples of 66 isolated Proteus were detected by plate-culture and Vitek auto microbe system, respectively, and the results were both consistent with the report from the samples donors the third Affiliated Hospital of Sun Yat-Sen University and the first Affiliated Hospital of Jinan University, indicating that the authenticity of those Proteus strains were proved.There pairs of primers for conventional PCRs were designed by taking the aptD and tuf genes as objective sequences to amplify the corresponding genes of 3 references Proteus, 66 isolated sample Proteus and 13 non-Proteus strains under the various PCR conditions. Results showed that 3 references Proteus and 66 isolated sample Proteus strains were all positive whereas 13 non-Proteus strains negative, which were exact consistent with the results detected by the conventional methods. However, the newly PCR methods took only 6-8 h, and appeared advantages of rapidity, concise, sensitivity and specificity. One specific primer specified for SYBR Green I fluorescent real-time PCR, based on atpD gene, were designed for rapid detection of Proteus. The tested results were exact same as the previous conventional PCR methods. However, the time spent for this detection was only 1-2 h, much more quick, specific, sensitive, simple than the conventional methods.Sequences of 8 bands of aptD genes tested results showed that there are no such sequences of Proteus aptD genes in the existed GenBank. Therefore, the sequences of Proteus aptD genes tested in this study are the first report in the world.In conclusion, the three conventional PCR methods and one specific fluorescent real-time PCR method are alternative reliable and rapid method applied for the rapid detection and identification of Proteus in food.