Research On Multiplex PCR Detection Method Of The Real Property Of Milk And Dairy Products

Real-Time PCR is used for detecting bovine,ovine,soybean,grain and plant derived components in this study.The establishment of multiplex RT-PCR detection method becomes the detection technology of high throughput screening and quick test.The main contents and results of article included:(1)DNA extraction methods.The establishment of stable DNA extraction methods for milk and dairy products by comparing phenol chloroform Method,Genomic DNA KIT and Beads method.Through the comparison of three different DNA extraction methods including Phenol chloroform Method,Genomic DNA KIT and Beads method,we choose the improved Genomic DNA KIT as the prior method for DNA extraction from milk and dairy products,DNA concentration and purity of extracted DNA from diary samples meet the requirements of the subsequent PCR test.(2)The establishment of RT-PCR system.We designed the specific primers and probes for the target organism.Stable RT-PCR system was found for the detection of bovine,ovine,soybean,grain-derived ingredients and plant-derived ingredients.The primers and probes designed could only detect target DNA fragments of each species.The PCR system for bovine could only detect bovine-specific DNA fragments and the system for ovine can only detect ovine-specific DNA fragments.There were no cross reaction between target species with non-target species.(3)Multiplex RT-PCR system.We further investigated the multiplex RT-PCR method for the simultaneous detection of bovine,ovine and soybean,plant-derived and grain-derived ingredients.(4)Specificity and sensitivity test.The specificity tests were conducted with the target species DNA and non-target DNA.Meanwhile,the sensitivity tests were conducted with ten-fold serial dilutions of target DNA and different concentration of target samples.For bovine,ovine and soybean,the detection threshold was 0.1 ng for target DNA and 0.1 %(w/w)for the simulation samples.For grain-derived ingredients or plant-derived ingredients,the detection threshold was 0.1 ng for target DNA and 0.5 %(w/w)for the simulation samples.(5)Market samples test.Established RT-PCR system was further applied to the detection of 65 market dairy products to verify the application ability of the methods.65 samples were collected in this study,most of them were milk powder.Bovine-derived ingredients were detected in three goat milk products and plant-derived ingredients were detected in five milk products.The qualitative real-time PCR system for detecting bovine,ovine,soybean,grain-derived ingredient and plant-derived ingredient were established in this study.The method was universal,specific,sensitive and reliable.It can be used to detect adulteration in milk and dairy products.It provides the technical support for the screening of adulteration so as to protect the benefit of consumers.Meanwhile,the method can be further applied to commercial kit,which means great social and economic benefits and efficiency.