A Multiplex PCR Technology For Meat Source Identification

Objective:Currently,the national standard method for meat source detection is based on single PCR and fluorescent PCR.However,the ingredients of the tested food are not clear and inefficient single-checking for non-directional screening is difficult to meet the needs of food safety testing.In this study,the mitochondrial DNA from eleven kinds of meat sources,including dogs,chickens,cattle,pigs,horses,donkeys,foxes,rabbits,rats,ducks,and sheep,is used as detection targets.A low-cost,high-sensitivity multiplex PCR system for detecting these 11 meat sources was constructed by establishing a multiplex PCR technique mediated by "Universal Primers"(UP-M-PCR),and corresponding kits for detecting meat products in the market were developed.Methods:1.Design and screening of primers:Primers in this study include universal primer,specific reverse primers,forward primers and reverse primers(the 5' end of the specific reverse primers are connected with a universal primer).The polymorphic sites of each tested meat source were determined by comparing and analyzing the genetic database data,and the forward primers and specific reverse primers of each meat source variety were designed.The Tm and amplification efficiency of the each pair of multiplex primers designed and screened are required to be as consistent as possible.The principle of universal primer design requires that the universal primers did not produce amplification products with mtDNA of these species in the process of PCR amplification.2.Construct a plasmid template to screen universal primer with the highest sensitivity:The three universal primers screened specifically are connected to both ends of the 16S rRNA gene partial sequence of the meat source by PCR reaction.The base sequence containing the tniversal primer was inserted into the plasmid vector.The extracted DNA of the positive plasmid was diluted as a template to detect the sensitivity of each universal primer.3.Construction of multiplex PCR system:The universal prime with the highest sensitivity among the three universal primers is connected to 5' end of all specific reverse primers to form corresponding reverse primers.Firstly,the forward primers and the reverse primers with the universal primer are compared and screened with each other.The specificity,sensitivity and amplification efficiency of 11 different meat source primers and multiplex PCR systems were verified by various PCR experiments.The multiplex PCR systems herein were subjected to a touchdown PCR method using a high annealing temperature of 71oCfor 10 cycles and a low annealing temperature of 60? for 25 cycles.The optimized multiplex PCR systems were tested on commercially available meat source foods,and verified the applicability of the multiplex PCR detection method.The accuracy of positive results was confirmed by sequencing.4.Detection of amplification products:All PCR products were analyzed and confirmed by gel electrophoresis.Results:A method for screening universal primers for meat nucleic acid detection was established,including universal primer-specific identification,and detection of universal primer sensitivity by construction of recombinant plasmids.Three universal primers were successfully screened,and a universal primer with the highest sensitivity(The sensitivity reached 100 copies)was used to join multiple specific reverse primers for constructing multiple detection systems.A 4-plex PCR mediated by universal primer for rats,foxes,ducks and mutton was constructed,and the detection sensitivity reached 0.05 ng/?L.The detection system can be applied to the identification of mutton adulteration in marketed meat products.By reducing the multiple primer concentration(multiple primer concentrations can be reduced to 0.00132?M),the detection throughput of this detection technique is effectively improved.An 8-plex PCR detection system for the identification of dogs,chickens,cattle,pigs,horses,donkeys,foxes and rabbits was successfully constructed.The sensitivity of the detection system can be as high as 0.05 ng/?L.It can be used to distinguish genuine and sham meat products for commercial meat products.Conclusion:The "universal primer" mediated multiplex PCR technique can be used for meat source identification,and the construction of two multiplex PCR systems mediated by universal primer could rapid identify 11 different meat sources in meat products.The method is simple,low-cost,fast and efficient,and its specificity and sensitivity can be used for the identification and detection of mixed meat products.