Development Of Polymerase Chain Reaction-based Techniques To Detect Aduleration Of Goat Milk With Bovine Milk

Milk and dairy products are rich in nutrition and are important basic food for human beings.However,a common phenomenon of fraud happens frequently,people mixed with cheaper bovine milk in goat dairy products to deceive consumers.In order to maintain market fairness and promote the healthy development of the dairy industry,it is very important for the detection of adulteration of dairy products.Based on the differences of the DNA between bovine and goat milk,two pairs of specific primers were targeted at mitochondrial 12S rRNA genes.Research and establishment of several rapid,low-cost PCR detection methods for differentiating bovine and goat milk.The following are the research contents and results:The DNA extraction methods and duplex PCR assay was developed of bovine and goat milk in this study.Compared to the DNA extraction of the Organic solvent method,the Rapid DNA kit method and the Magnetic-bead method from fresh milk,this technique was used the extracted DNA as a template and specific primers to amplified the target DNA fragments of 256 bp and 326 bp for bovine and goat,respectively.Through the identification of the completeness of the DNA fragments by the three methods described above and the the electrophoresis results of PCR amplification showed that the Organic solvent method had low efficiency of DNA extraction in milk.The purity of DNA obtained by the Rapid DNA kit method was preferable,but it had a poor repeatability.The DNA extracted by the Magnetic-bead method had the best purity,high extraction efficiency and good reproducibility.The concentration of DNA extracted from milk was determined indirectly by using the dyestuff method.The concentration of goat and bovine DNA was 2.132μg/mL and 1.186μg/mL,respectively.The results of the duplex PCR displayed that the Rapid DNA kit method was able to detect 10%（vol/vol）bovine milk adulterated in goat milk,but the Magnetic-bead method could be detected the proportion of bovine milk incorporated as low as 1%.Therefore,the subsequent experimental studies were carried out using the Magnetic-bead method to extract DNA from the samples.Investigated the detection technique of bovine milk adulterated in goat milk by single real-time PCR.In order to overcome the limitation of time-consuming,low-flux and contamination in the electrophoresis analysis of conventional PCR,a real-time PCR method based on DNA-binding dye（SYBR Green I）was established to identify the adulteration of bovine milk ingredients in goat dairy products.The template DNA of bovine and goat were added to their respective primers（153 bp and 118 bp）for real-time PCR amplification.The Ct value of the amplification curve was used to differentiate the bovine and goat milk,and the PCR-related parameters were optimized.After gradient dilution amplification and linear fitting,the reactions were able to reach 10^-5 and 10^-4,the R² was 0.995and 0.996 for bovine and goat milk DNA,which had good amplification efficiency and repeatability.The results indicated that the proposed method can detect the 2.5%adulteration level of bovine milk in fresh goat milk,and its practical application was demonstrated with the identification of ingredients derived from bovine and goat in 11 commercial dairy products.A duplex real-time PCR method based on EvaGreen for the identification of bovine milk adulterated in goat milk,the aim of this method was to further increase the flux,save the reagent and reduce the cost of detection.Two pairs of specific primers were added simultaneously to the template DNA and optimized the conditions.The composition of the goat and bovine milk was discriminated by the different Tm value of the DNA melting curve.Melting curve analysis revealed that the Tm values of the primers of bovine and goat were 79°C and82°C.By comparing SYBR Green I dye with EvaGreen dye,EvaGreen dye could significantly improve the resolution of the melting curve.Different proportions of bovine and goat milk were mixed and results showed that the technique allowed the detection limit of 0.05%for bovines’milk in goats’milk and the proposed method was successfully applied to 12 commercial dairy products.Explored the detection of milk DNA by real-time PCR with the dye of Ruthenium Complex.SYBR Green I and EvaGreen were imported commercial reagents with patent protection,unclear structure information and costly.Therefore,Ru complex DNA-binding dyes were used in real-time PCR assays for the preliminary exploration.The experimental results showed that a PCR amplification curve for quantification can be obtained by real-time PCR to monitor the fluorescence signal changes by the ruthenium complex dye.According to the serial dilution and fitting,the DNA had a good linear relationship under Ru action.The R² were 0.974 and 0.986 for bovine and goat milk DNA.And the detection sensitivity was equivalent to that of SYBR Green I and EvaGreen.It was expected to be developed as a new detection reagent suitable for the application of real-time PCR.Through the conventional PCR,single and duplex real-time PCR in dairy adulteration,and explored thde real-time PCR detection with the ruthenium complex dye.Compared with the methods using protein,fat and so on as the detection target,several PCR methods based on DNA were established in this study,which had the advantages of reliable results,easy operation and high throughput.It could provide technical reference for adulteration inspection method of dairy products.In addition,the established method had certain versatility and could be further promoted for the authenticity ddetecting of foods such as meat products,aquatic products and oil products,etc.Overall,this techinque could lay the foundation for improving the food labeling system and providing an effective detection means for food supervision and management to counteract fraud.