Polymerase Chain Reaction For The Rapid Detection Of Food-borne Salmonella

ABSTRACT: Salmonella continues to be a major food-borne pathogen inanimals and humans, and is a leading cause of food-borne outbreaks and infections inmany countries. Outbreaks are reported frequently, implicating different kinds of foodcontaminated with Salmonella. A majority of cases of human salmonellasis are due tothe consumption of raw or undercooked foods, including meat, eggs and dairyproducts. Because currently effective methods for inactivating Salmonella in foods arestill being researched, detection system plays a significant role in preventing thedissemination of contaminated food products. Therefore, sensitive and rapid methodsare required for the detection of food-born Salmonella to ensure food safety. In thispaper, the reaction system and conditions of real-time PCR based on DNA wereoptimized and the specificity, sensitivity, repeatability and food additive tests werecarried out to analyze the availability and reliability of new assay. At the same time,the effects of different heat treatments on the degradation of Salmonella DNA wereresearched. The effects of dead bacteria on the results of real-time PCR were alsostudied. At last the RNA-based real-time reverse-transcriptase PCR were establishedand preliminary applied to the detection of artificially contaminated foods. Theaffection of dead bacteria on real-time RT-PCR were validated.Results showed that:(1) The optimal reaction conditions of real-time PCR were42°C/5min,95°C/5s, then95°C/5s,67°C/34s for40cycles; followed by meltingcurve analysis. The total volume of reaction system was20μl, of which the forwardprimer and reverse primer (1μM) was2μl respectively. The newly designed real-timePCR was specific, repeatable and sensitive. It did not require pre-enrichment reducingthe testing time greatly, the experimental operation process could be completed in4hours. Various foods artificially contaminated with Salmonella were detected usingthe new method. The sensitivity of the method was sufficient to detect1.9×10~2cfu/g,4.5×10~2cfu/g and1.5×10~2cfu/ml of salmonella in fresh pork, prepared chicken andmilk respectively. Both the qualitative and quantitative results of detecting artificiallycontaminated foods were in accordance with the traditional method and it was proved to be a simple method for the quantitative detection of food-born salmonella.(2) Thetreatments of65°C/20min,85°C/30min, boiling-for-3min,121°C/15min whichwere commonly used in the processing and consumption of food can completelyinactivate the Salmonella. The higher heating temperature caused greater destructiveto Salmonella DNA. However, the bacterial DNA did not disintegrate completely afterheat treatments, In a very long period, there were still a large number ofslowly-degradation gene fragments in death cells.(3) The remaining genomic DNA orDNA fragments in dead Salmonella had a significant effect on the testing results ofreal-time PCR. Results were positive for dead Salmonella suggesting that theDNA-based real-time PCR could not distinguish dead from viable bacteria effectively.The method is suitable for fresh food detection instead of containing dead bacteriasamples such as processed meat products, ready-to-eat foods.(4) The optimal reactionconditions of real-time RT-PCR were42°C/5min,95°C/5s; then95°C/5s,67°C/34s for40cycles followed by melting curve analysis. The total volume ofreaction system was25μl, of which the forward primer and reverse primer (1μM) was4μl respectively. The newly designed real-time RT-PCR did not requirepre-enrichment reducing the testing time remarkably, and could be completed in4hours. Various foods artificially contaminated with Salmonella were detected usingthe new method, The sensitivity of the method was sufficient to detect1.9×10~2cfu/g,4.5×10~3cfu/g and1.5×10~3cfu/ml of salmonella in fresh pork, prepared chicken andmilk respectively. They were lower than the sensitivity of real-time PCR. Thequantitative results were not as good as the results of real-time PCR. But its biggestadvantage is that can distinguish between dead and viable cells to compensate for thelack of real-time PCR. Therefore, it is more suitable for the qualitative detection offood-borne Salmonella in the samples.