A PCR-mtDNA Approach For The Identification Of Lamb, Pork, Chicken And Duck

In recent years, with the increasing demand of lamb, counterfeited mutton, especiallyby pork, chicken or duck, begins to emerge in the market, not only disrupting the marketorder, but also endangering the health of people. Considering that there’s no standard tofollow in the operation of processing lamb, we should first develop an unified detectingmethod in order to reduce the crime of counterfeiting mutton.We aim to establish a one-time multiplex PCR approach to quickly detect pork,chicken or duck composed in lamb. With its small size,conservative structure, largenumber of copies,the stability against heat or degradation, matrilineal inheritance and fastevolutionary rate, mitochondrial DNA is widely used to provide sequence information forthe evolutionary analysis in or among species, or the identification of animal-derivedingredients in foodstuff or feedstuff.Online BLAST of NCBI and DNAMAN software were applied to analyseconservative regions and variable regions of mitochondrial DNA of sheep,pork,chickenand duck. Primer5.0and Oligo6.0software were applied to design more than30primerpairs which were consequently tested one by one. Four pairs of primers were at last chosen.Each of the four pairs of primers is designed to detect species-specified mitochondrialDNA fragment in one of the four species individually. The amplified fragments aredistinguished from each other in length differences, which can be detected byelectrophoresis. A multiplex PCR approach was established to hold the four pairs ofprimers at one time because similar annealing temperature and reaction system (Mg2+concentration has little effect on the reaction). In order to ensure the accuracy of the PCRreaction, large numbers of tests were made to ensure that the four pairs of primers and thefour kinds of DNA templates wouldn’t have mutual interference. At last the system andconditions of multiplex PCR were determined.The detecting method contains3major steps.(1) extracting genomic DNA fromtissues using a genomic DNA extraction kit.(2) PCR amplification.(3) electrophoresisdetection of amplified product, with the DNA Marker or standard samples to determine thecomponents in the unkown sample.In order to estimate the sensitivity and accuracy of the multiplex method, a series ofgradiently diluted genome DNA templates were amplified, and templates diluted as manyas1000times were detected. Template of each species gradiently diluted by other genome DNA were amplified, and detective sensitivity is not affected. Meat samples mixed withcorn flour or fish as many as100times before extracting DNA were detected. Boiled meathave slightly lower sensitivity because of unsignificant degradation of mitochondrial DNA.A mixture of a variety of meat tissue was repeatedly tested to verify the stability andreliability of the method.This method has been applied for patent, the national invention patent applicationacceptance number:201310123666.1.