Screening Of Target Sequences And Establishment Of Rapid And Accurate Detection Method For Identification Of Common Carnivore-derived Ingredients In Meat Products

With the increasing demand for meat,the safety of meat products has attracted extensive attention.Some unscrupulous merchants even use the meat of cats,dogs,foxes,minks and other common carnivores for adulteration and sale.According to reports,some carnivores such as cats,dogs and minks are at risk of infecting and spreading the Severe acute respiratory syndrome coronavirus-2(SARS-Co V-2),and are hosts of a variety of zoonotic diseases.Eating these meats can easily lead to the spread of diseases,which brings hidden dangers to people’s health.At present,a variety of identification methods of animal-derived ingredients based on species-specific DNA sequences have been established.Most of these methods take mitochondrial DNA as the target,which is easy to produce false negative results due to the variation of primer binding region of different varieties or individuals.Moreover,there methods often need to use multiple pairs of primers at the same time,and the interaction between primers will adversely affect the efficiency and accuracy of detection.It is more difficult to simultaneously detect various carnivores on the spot.Therefore,this study intends to screen cat-,dog-,fox-and mink-specific target sequences on the nuclear genome.By combining universal primers with RPA and real-time PCR,we aim to develop a rapid on-site screening method and an accurate laboratory detection method of common inedible carnivore-derived ingredients.The main results are as follows:(1)Screening of target sequences for the detection of cat-,dog-,fox-and mink-derived ingredients: Through alignment analysis of nuclear genome sequences of multiple species,a DNA fragment(NC＿051821.1)from dog’s nuclear genome was screened out as a target sequence for identifying and distinguishing cat,dog,fox and mink.The homology of the target sequences of cat,dog,fox and mink is not less than80%,and the sequence lengths are 339 bp,335 bp,335 bp and 332 bp,respectively.While there are no homologous sequences in other 40 non-target species or there are homologous sequences with a very low homology and a fragment length of less than 200 bp.By using Clustal W and MEGA7 software,we found that the target sequences of cat,dog,fox and mink had higher sequence differences as a whole compared with the homologous sequences of other species.Only for the target sequences of cat,dog,fox and mink,the two ends of four target sequences are well conserved,but there are obvious base differences inside.These characteristics indicate that the target sequences of cat,dog,fox and mink are relatively conservative for target species(cat,dog,fox and mink),and have good specificity for non-target species,which can be used to identify cat,dog,fox and mink.(2)Establishment of a rapid screening method for cat-,dog-,fox-and mink-derived ingredients.The universal primers of RPA were designed in the conserved regions of the target sequences of cat,dog,fox and mink,and the reaction system and conditions of RPA were optimized.The reaction can be carried out at a constant temperature(42℃)and only takes 30 min to complete.The RPA products were analyzed by agarose gel electrophoresis,and it was found that the target bands were amplified only in the positive samples of cat,dog,fox and mink,however,there was no amplification products in common animal species from meat products such as cattle,sheep,donkey,pig and chicken,indicating that the established RPA method can be used for preliminary screening of cat-,dog-,fox-and mink-derived ingredients in meat products.As long as the sample contains cat,dog,fox and mink fractions,it can be detected,but the 4 target species could not be distinguished.(3)Establishment of accurate laboratory detection methods for cat-,dog-,foxand mink-derived ingredients.Universal primers of real-time PCR were designed in the conserved regions of the target sequences of cat,dog,fox and mink,and species-specific Taq Man probes were designed in the hypervariable regions of the target sequences of cat,dog,fox and mink,respectively.The specificity of primers and probes was assessed in 21 species via simplex real-time PCR.The results proved that the primers and probes have good specificity.The reaction conditions and reaction system of multiplex real-time PCR were optimized,and the optimal ratio of specific Taq Man probes for cat,dog,fox and mink was 6:3:8:2.A universal primer multiplex real-time PCR(UP-M-rt PCR)method for the accurate identification and differentiation of cat-,dog-,fox-and mink-derived ingredients in meat products was established.The absolute detection limit of this method was 0.05 ng/μL,the relative detection limit was 0.05%(w/w).The method was used to detect 6 multispecies meat mixtures with different adulteration ratios,and the detection results were completely consistent with the actual adulterated components,which proved that the method has excellent accuracy.In summary,this study screened the target sequences of cat,dog,fox and mink on the nuclear genome.A rapid RPA screening method and an accurate UP-M-rt PCR laboratory detection method were established for cat-,dog-,fox-and mink-derived ingredients,which can provide effective technical support for regulatory authorities.