A Study Of Detecting Microorganism In Food By Real-time PCR Method With Universal Primers And Probe

Microbial contamination could be seen anywhere, the food hygiene indicators stipulatedthe detection method of the total number of colonies, mold and yeast. However, the methodhad the disadvantage of long operation steps and delaying the test results. A rapid real-timePCR method for detecting microorganism in food by real-time PCR method was developed.The probe and primer was designed according to the genome sequences of bacteria, yeast andmold respectively. The method was not only tested with its specificity, sensitivity andreproducibility, but also applied to the detection of food samples. At the same time, themethod of optimizing template DNA extraction was also developed.We established the method of bacterial and fungal DNA rapid extraction by integratinglysis solution, boiling and liquid nitrogen grinding, which was suitable for real time PCRtechnology. We optimized the extracting concentration and demonstrated the effectiveness ofthe law through the sensitivity, reproducibility and applying to the detection of food samples.The result of optimizing extracting concentration showed that the best extractingconcentration for bacteria and mold were triton-X 100 4% and tween-20 0.5%, benzylchloride 40% and SDS 1% respectively.The strain of sensitivity experiments containedgram-positive bacteria such as Listeria monocytogenes, gram-negative bacteria such asSalmonella typhimurium, Saccharomyces cerevisiae and Aspergillus flavus. The resultsshowed a high sensitivity of detection which could detect 460 cfu/m L of Salmonellatyphimurium, 540cfu/m L of Listeria monocytogenes, 580 cfu/m L of Salmonella typhimurium,600cfu/m L of Aspergillus flavus. The strain of reproducibility experiments containedSalmonella typhimurium and Aspergillus flavus. The method also showed a highreproducibility, the variations(CVs) of Salmonella typhimurium were respectively0.58-1.15% and 0.27-0.87% with the sample and between tests. And the CVs ofAspergillus flavus were respectively 0.45-1.57% and 0.78-0.99% with the sample andbetween tests. The sample can be checked out the positive when the strain was put into thesamples.The probe and primer were designed according to the 16 S r RNA genome sequences ofbacteria, this chapter demonstrated the effectiveness of the probe and primer through thespecificity, sensitivity, reproducibility and applying to the detection of food samples. Theresults showed that the real-time PCR assay protocol showed a good specificity by detectingonly bacterium and was not affected by yeast and mold, the sensitivity of detectingEscherichia coli O157 and Staphylococcus aureus were 420 cfu/m L and 550 cfu/m Lrespectively. The reproducibility result showed that the Escherichia coli O157’s CVs wererespectively 0.56-1.03% and 0.54-1.01% with the sample and between tests, and theStaphylococcus aureus’ s CVs were respectively 0.75~1.09% and 0.58-1.29% with the sampleand between tests.The samples which had concentration of 5 cfu/25 g could be checked out thepositive after 8h when Escherichia coli O157 was put into the samples. However, it only took6 h for the samples which had concentration of 10 cfu/25 g and 15 cfu/25 g. The samples whichhad concentration of 5 cfu/25 g and 10 cfu/25 g could be checked out the positive after 8hwhen Staphylococcus aureus was put into the samples, as for concentration of 15 cfu/25 g,itonly took 6h to check out the positive.According to the 26 S r RNA genome sequences of yeast and the 28 S r RNA genomesequences of mold, we designed the probe and primer of yeast and mold respectively. theeffectiveness of the probe and primer were demonstrated through the specificity, sensitivity,reproducibility and applying to the detection of food samples. The result of yeast specificityshowed a good specificity by detecting only yeast and was not affected by bacterium andmold, and so did the mold specificity. The sensitivity of detecting Saccharomyces cerevisiaeand Aspergillus oryzae were 570 cfu/m L and 620 cfu/m L respectively. The reproducibilityresult showed that the Saccharomyces cerevisiae’s CVs were respectively 0.62-0.81% and0.43-0.77% with the sample and between tests, and the Aspergillus oryzae’s CVs wererespectively 0.75-1.03% and 0.68-1.13% with the sample and between tests.The sampleswhich had concentration of 5 cfu/25 g and 10 cfu/25 g could be checked out the positive after12 h when Pichia pastoris was put into the samples, it only took 10 h for the samples whichhad concentration of 20 cfu/25 g, and so did Aspergillus oryzae.In short, the established real-time PCR assay in this study has sufficient specificity, highsensitivity and good reproducibility, and it has the advantage of speediness, simpleness,cheapness which makes it a very good detection method.