Development Of DNA-based Identification Technologies For Mutton Adulteration

In recent years,there have been an increasing number of adulterations of meat and meat products.In China,mutton are among the most favorite foods which are often adulaterated with other low-cost animal meat such as chicken,pork or duck meat.There is an old Chinese saying"cry up wine and sell vinegar"reflecting this phenomenon.The European horse meat scandal in 2013 caught the attention of the public and drawn great attention of the related government departments.At present,mutton adulteration detection methods rely principally on nucleic acid based qualitative PCR.Even digital PCR that has been developed recently which could achieve quantitative detection.However,it requires expensive reagents and instruments,complicated and time-consuming operating steps.Therefore,it is urgent to develop a rapid method for detection of mutton adulteration.In this study,A simple lysis solution for quick DNA extraction was developed and a fluorescence quantitative PCR method for mutton detection based on mitochondrial or nuclear single-copy gene was established respectively.In addition,a Recombinant polymerase amplification(RPA)-based lateral flow strip was also developed for mutton quickly identification.This study includes the following contents:(1)The effects of different lysis solution on meat DNA extraction were evaluated and the results indicated that NaOH solution had the best effect.This lysis solution could extract meat DNA within 5minutes,requiring no grinding and centrifugation procedures.Besides,it is easy to prepare this low-cost solution which can be stored for a long time.Therefore,this solution provided preconditions for establishing a fast method for mutton adulteration detection.(2)A real-time PCR method for quantitatively determining mutton adulteration was established based on the reference mitochondrial gene and mutton-specific mitochondrial gene.Mutton and pork were mixed with different ratio to prepare spiked adulterated samples.Taking the mutton content as X-axis,the Ct ratio(Ct value of the standard sample amplified by mutton-specific gene/Ct value of the standard sample amplified by reference gene)as Y-axis,the standard curves could be plotted and the regression equation were established as Y=-0.3166X+1.2113 with R~2=0.9929,indicating a good linearity.When this method was used to detect simulated samples,the average recovery is108.74%indicating that the method proposed in this study could be used for quantitative detection of adulteration of mutton with pork.(3)Nuclear single-copy reference gene and mutton-specific gene based real time PCR was developed for quantitatively mutton adulteration detection.Reference gene was used to correct the contents of the mutton species specific gene,and standard curves was established based on the logarithm of the initial template concentration and the Ct value of the standard DNA solution amplified by PCR.The regression equation were established as Y=-3.9416X+27.954 and Y=-3.5507X+28.844,with the coefficients R2=0.9999 and R2=0.9993,respectively.According to the simulated sample detection results,the average recoveries for mutton-pork,mutton-duck and mutton-chicken mixtures were 89.317%,95.11%and 107.15%,respectively.This method could identify the concentrations of mutton in adulterated sample within 3h.The simple,rapid,sensitive and low-cost method show broad prospects for quantitative detection of adulteration of animal components.(4)Combined with Recombinant polymerase amplification(RPA),a colloidal gold based lateral flow dipsticks were developed for visual identification of mutton.This approach could achieve qualitative detection of mutton within the range of 20 ng/μL to 2 pg/μL and the whole detection could be finished within 30 min with visual results displayed.This method is simple,sensitive and rapid which can be used for onsite detection with its portable characteristics.