Rapid Identification Of The Animal Origin Components Using Cross Priming Amplification

Nowadays,various reports of meat adulteration have strengthened the awareness of both consumers and government,and the problem of meat adulteration have accounted for a greater proportion in animal-derived food safety issues.The existing laboratory testing method can’t meet the needs of market testing.1,Comparing mt DNA sequences of the duck,sheep,pigs,cattle,chickens,and select specificity fragments in duck D-loop gene,in sheep Cytb gene and in pork D-loop gene respectively;2,According to the principle of CPA reaction,three methods were used to design and screen out CPA primers and probes on the specific fragment;3,For the specific fragment,PCR primers were designed and the plasmid containing the target fragment was prepared;4,The optimum ranges of all factors were obtained by optimization experiments: the concentration range of cross-primer was between 0.6 μmol·L-1~0.8 μmol·L-1,the concentration range of labeled primer was between0.4 μmol·L-1~0.6 μmol·L-1,the concentration range of magnesium ion was between 4 mmol·L-1~8 mmol·L-1,the concentration of betaine had no significant effect on the amplification,and it could meet the requirement of CPA amplification when the reaction time reached 45 ~ 60min;5,In the specific experiments,the three component detection methods for animal source were all have good detection specificity.Duck source CPA detection method had no cross reaction with the source component of sheep,cattle,chicken and pig;Sheep source CPA detection method had no cross reaction with the source component of duck,cattle,chicken and pig;Pig source CPA detection method had no cross reaction with the source component of sheep,cattle,chicken and duck.6,The detection limit of duck source component CPA detection method for single duck source component was 0.1 ng·μL-1,and it could reach 1% for mixed samples.The detection limit of sheep source component CPA detection method for single source component could reach 1000 cpoies·μL-1,and it could similarly reach the detection limit of 1% for mixed mutton products.The detection limit of pig source component CPA detection method for single pig source was only 1 ng·μL-1,and it was only 10 for mixed pork.7,CPA test method in the repetitive experiments show good stability;The nucleic acid method for single animal-derived component detection in mixed meat products owned specific advantages.With perfect detection specificity,simplified operation,and low-dependency of equipment,this method would be a promising detection method for further research.