Identification Of Goat,Sheep,Bovine And Water Buffalo-Derived Ingredients In Dairy Products By PCR-HRM

Identification of adulterated milk and dairy products is an important challenge for food quality and safety control.In recent years,PCR,especially multiplex PCR,has provided an important method for rapid identification of animal origin of dairy products.However,most of them use electrophoresis,multicolor fluorescence labeling or sequencing to achieve multiplex detection,which are laborious and expensive.In this paper,a series of PCR methods were developed by using HRM technology to identify goat,sheep,bovine and buffalo in dairy and dairy products accurately,rapidly and cost effectively.The contents and results were detailed as follows:(1)Duplex PCR-HRM method for identification of bovine milk components in buffalo milk.By comparing the mitochondrion genome sequences of bovine and buffalo,the species-specific primers of bovine and buffalo were designed respectively.The results showed that the Tm values of the amplified products of PCR were 77℃ and 81℃,respectively.There was overlap and interference using the traditional melting curve to detect of bovine and buffalo mitochondrion genome DNA at the same time.However,in HRM analysis,the melting peaks of buffalo and buffalo were completely separated,which indicated that HRM could distinguish the amplified products with little difference in Tm more easily and effectively.After optimized the double PCR-HRM reaction system,HRM was proven capable of effectively identifying the presence of bovine DNA down to 1% and also of quantifying the ratio of bovine in water buffalo milk by linear fitting the values of melting peak height and the logarithm of adulterated proportion.(2)Multiplex PCR-HRM method for identification of ingredients of goat,sheep,bovine and buffalo in dairy products using specific primers.For further improve the detection throughput,a method for simultaneous detection of four animal-derived ingredients in dairy products was established by screening and adjusting primers on the basis of previous work.The results of PCR-HRM showed that the melting temperatures of goat,sheep,bovine and buffalo amplicons in the quadruplex PCR-HRM system were 86.7,77.7,83.7 and 80.2,respectively.There was no overlap between the melting peaks.The results of specificity,sensitivity and different mixtures of samples showed that the method has good specificity and sensitivity,and there is no interference between different species.The detection limit of this method is 2pg for sheep DNA and 0.2pg for goat,bovine and buffalo DNA.It can also realize rapid and accurate identification and detection of goat,sheep,bovine and buffalo-derived ingredients in milk and dairy products.(3)Duplex PCR-HRM was developed for identification of bovine milk ingredients in goat milk.In the above work,PCR-HRM can effectively distinguish the amplified products with small Tm difference,but the Tm difference still needs to be >2℃.In this study,HRM technology can provide more abundant melting curve information and realize the simultaneous detection of goat and bovine PCR products with Tm difference less than 1℃.The results showed that the Tm of goat and bovine PCR amplicons were 80.0℃ and 80.3℃,respectively.It was difficult to identify the ingredients of goat and bovine simultaneously because of overlapping the melting peaks of HRM.However,the application of HRM curve shape analysis allowed discrimination of goat and bovine simultaneously.In addition,the method can also quantitatively detect the adulteration ratio of mixed samples,and the detection limit of adulteration of bovine DNA to goat DNA reaches 0.1%.Therefore,PCR-HRM can accurately identify the multiplex PCR products with Tm difference less than 1℃.(4)PCR-HRM method detection of source components of goat,sheep,bovine and buffalo using universal primers.In the above multiple PCR reactions,multiple pairs of specific primers are needed to identify multiple species at the same time.The existence of multiple pairs of primers can easily lead to non-specific amplification,and the amplification efficiency among primers is inconsistent and needs repeated optimization.In order to overcome this limitation,a pair of universal primers were designed to amplify the target genes of goat,sheep,bovine and buffalo simultaneously in a single reaction vessel.The results showed that the primer pair had good universality and specificity.Meanwhile,each amplified sequence could produce a unique HRM profile and the four species could be rapidly identify through clearly-distinguishable melting graphs.In addition,the method can distinguish all 15 possible mixtures accurately and effectively,and can quantify the adulteration ratio of bovine in buffalo and goat.Therefore,PCR-HRM technology can realize the rapid identification of goat,sheep,bovine and buffalo-derived ingredients in milk and dairy products.