Development Of Methods For Detection Of Food Source Chicken Ingredients Based On Loop-mediated Isothermal Amplification

Meat adulteration is one of the most important factors affecting food quality and safety,and nucleic acid detection methods play an important role in it.The Loopmediated isothermal amplification(LAMP)technique has received much attention and is widely used in the field of nucleic acid detection because of its easy operation and low cost.In this study,the construction of a LAMP method based on SYBR dye and molecular beacons for the detection of food-derived chicken components was carried out,and the construction of LAMP immunochromatographic test strips for chickenderived components was carried out on this basis:(1)The LAMP primers were designed and optimized to target the cytb gene,dloop gene,12 S r RNA gene and 16 S r RNA gene in chicken mitochondrial DNA,respectively,and on this basis,a LAMP assay for chicken meat components was established using SYBR dye as the signal system.The results showed that the LAMP method established with cytb-2 and 16-1 primers had good specificity and sensitivity,and could detect 10 pg and 1 pg of chicken DNA,respectively,and the detection limits of artificially simulated meat adulteration were 2.5% and 1%,respectively,which were more consistent with the q PCR-based method in terms of specificity.(2)The 16-1,d-loop-3 and cytb-2 molecular beacon probes were designed for chicken meat components,and on this basis,the LAMP assay based on molecular beacon probes was established by optimizing the conditions of molecular beacon probe concentration and reaction temperature.The results showed that under the conditions of DNA extraction by alkaline lysis method,the LAMP detection method based on molecular beacon probes with 16-1,d-loop-3 and cytb-2 primers could detect 300,30 and 300 pg of chicken DNA,respectively,and the detection limits of artificially simulated meat adulteration were 4%,1% and 4%,respectively;under the conditions of DNA extraction by genomic DNA extraction kit,the The molecular beacon probe LAMP assay based on 16-1,d-loop-3 and cytb-2 primers could detect 3,3 and 30 pg of chicken DNA,respectively,and the detection limits of artificially simulated meat adulteration were 0.5%,0.25% and 1%,respectively.(3)Based on d-loop-3 primers,Bio-LF primers suitable for immunochromatographic test paper platform were designed,combined with LAMP amplification,and colloidal gold was used as the detection signal to initially construct the LAMP immunochromatographic test paper for chicken-derived components,when the LAMP product addition amount was 3 μL,the gold standard anti-FAM antibody addition amount was 10 When LAMP product was added at 3 μL,gold standard antiFAM antibody was added at 10 μL and Bio-LF was added at 1.2 μL,LAMP immunochromatographic test paper could detect 1 pg of chicken DNA under the condition of DNA extraction by genomic DNA extraction kit,and the detection limit of artificially simulated meat adulteration was 0.12%;LAMP immunochromatographic test paper could detect 10 pg of chicken DNA under the condition of DNA extraction by alkaline lysis method,and the detection limit of artificially simulated meat adulteration was 0.12%.The limit of detection for artificially simulated meat adulteration was 0.5%.