The Establishment And Application Of PCR-Chromatography Strip Rapid Detection Method For Ingredients Of Animal Origin In Food And Feed

This study has established a PCR-chromatography strip rapid test method in the foods and feeds of donkey and dove origin.Extract the constituent of animal DNA first,and then design primer for species specific DNA sequences,and mark primers,The 5 'end of the two pairs of primers respectively be marked by fluorescein isothiocyanate(FITC)and biotin,finally interpretate results by chromatography dipsticks.By combine PCR with general immune chromatography test paper,achieved a simple,fast,low cost,and visual detection method for animal origin in foods and feeds.1.The specificity of primers:Take donkey and dove meat as positive samples,other animal and plant samples as negative samples.Amplificate these samples by donkey primers and dove primers tagged.Test these samples using method in this study.Results show that the primers with high specificity is effective in amplification of donkey and dove samples.2.Absolute sensitivity:Dilute the donkey and dove DNA 5 times,the concentration respective is 100ng/?L,20 ng/?L,4 ng/?L,0.8 ng/?L,0.16 ng/?L,0.032 ng/?L,0.0064 ng/?L,0.0013 ng/?L,0.0003 ng/?L,0.0001 ng/?L and 0.00002 ng/?L.Test the samples using method in this study.Results show that the absolute sensitivity of this method is 0.0013 ng/?L.3.Actual sensitivity : Take donkey and dove meat as positive samples and beef as sample of adulteration.Slice donkey meat,dove meat and beef,and then dry them at 60 ?.Grind them into powder and sieve powder with18 mesh.Blend thesamples to the quality percentage(W/W)of donkey(pigeon)meat powder to 100%,50%,25%,10%,5%,1%,0.1%,0.01%,0.001%,and then extract the genome DNA to DNA concentration 5 ng/?L.Test the samples using method in this study.Results show that this method for mixed samples detection with high sensitivity and the minimum detection limit is 0.01%.4.The actual sensitivity of processing samples:Process the donkey meat and beef,dove meat and beef samples with 100 ? high temperature 2 h and fried 5 min.Results show that the minimum detection limit decreased to 0.1%(w/w)after the high temperature and fry after processed.5.Stability:Test no handle 0.01% of donkey meat and beef,no handle 0.01% of dove meat and beef,0.1% high temperature mixed meats,0.1% fried mixed meats 20 times to test the minimum detection limit.Results showed that the strip's detection line is red in 20 times,and the results are all positive.The stability of minimum detection limit is good and detection rate is 100%.