| Animal-derived components are representative substances derived from animal species,which can be nucleic acids,proteins,lipids and small molecule species.In recent years,various kinds of adulteration and doping incidents of animal products emerged,such as the "horse meat scandal" in Europe,the "fake mutton incident" in China,and the global spread of mad cow disease caused by illegal addition of ruminant ingredients in feed,etc.These safety problems of livestock and poultry products seriously affect the order of meat market and the healthy development of animal husbandry.Qualitative and quantitative detection of animal-derived ingredients in food and feed is an important technical means to solve these problems.At present,PCR method based on mitochondrial DNA(mtDNA)is the main technology.However,due to the difference in the copy number of mtDNA between tissues and individuals and the high variability of bases,it is easy to produce false negative test results,and difficult to achieve accurate quantitative detection.Therefore,it is urgent to screen new species-specific DNA sequences for accurate detection of animal-derived components.In addition,the poor conservativeness or lack of multi-species shared internal reference also affects the accuracy of quantitative detection of livestock and poultry products.In this study,we used animal genome sequences and transcriptome informations to screen species-specific nuclear DNA sequences and conservative nuclear DNA sequences through comparative genomics analysis strategy,and established qualitative and quantitative detection methods for various animal-derived components in meat and feed.This will provide technical support to meet the urgent needs of animal product safety supervision departments for the systematic,diverse and rapid detection of animal-derived components.Screening of species-specific DNA sequences and analysis of their molecular characteristics.To obtain new species-specific DNA sequences,three local BLASTN were preformed aligned with Nt database,whole genomes of other species and genome of target species based on the exon sequences of species,using dynamic E-value combined with Identity% and Quary-cover% as screening parameters.And species-specific exons were screened from 19 species of 11 families according to the taxonomic status of species.After that,all species-specific exons were classified into complete exon specific sequence(CESS)and partial exon specific sequence(PESS).And their distribution,structure and sequence characteristics were analyzed.(a)Species-specific exons account for a very low proportion of total exons,and are independent of the size of chromosomes.(b)CESS is mainly distributed in the CDS region and contains many repetitive elements in the sequence.(c)Shorter exons with high GC content are more prone to complete deletion or increase of fragments,which is related to the repetitive sequence in the upstream and downstream of the sequence and the short repetitive sequence in the sequence.Finally,the primers were designed by using specific DNA sequences of each species,and the specificity of species-specific DNA sequences and primers were validated in 19 animal DNA samples by PCR.The results indicated that the selected species-specific DNA could be used as a target sequence for the detection of animal-derived components.Qualitative PCR detection of animal-derived components in meat products and animal-derived feeds based on species-specific DNA sequencesTo solve the problem of lack of target sequence in the detection of animal-derived components,as well as the insufficient inter-species specificity and intra-specific conservation of target sequences,a qualitative PCR method for detection of the components of bovine,sheep and goat,pig,chicken and duck was established based on the species-specific DNA sequences screened previously.Through on-line BLAST and ClustalW multi-sequence alignment analysis,each species-specific DNA sequence was aligned with the reference genomes of 53 representative species including 4 kingdoms,11 classes,21 orders,36 families and 47 genera,and the high inter-specific specificity and intra-specific conservation were verified.Based on the design of species-specific primers and optimization of reaction system and reaction conditions,a PCR detection method for 5 species with 5 pairs species-specific primers and same reaction procedure(annealing temperature is 60?)was established.This realized simultaneous detection of components of bovine,sheep and goat,pig,chicken and duck in meat.The results of assay in 19 species DNA showed that the established PCR method is highly specific,only had amplification products in target species,and no products in other species.The results of PCR amplification and sequencing validation in 44 different individual DNAs of each species indicated that these species-specific sequences are highly conservative within species.Sensitivity tests showed that the detection limit of this method was 0.5 ng DNA.Finally,the cooked meat products of beef,mutton,pork,chicken and duck meat were tested.And the results consistent with their meat source,which indicated that the method could be used for detection of the derived components of processed meat.Aiming at the feed-borne transmission of mad cow disease,the bovine specific TAF4 gene was screened.There were 18 bp fragments difference in the gene between bovine and caprine.A pair of universal primers were designed for this fragment,but the amplified products were different in size for bovine and caprine.The bovine(125 bp)and caprine(107 bp)derived components could be simultaneously detected and identified by a single PCR reaction,and showed high inter-species specificity in 23 species.Different breeds of bovine(cattle,yak,zebu,buffalo),caprine(sheep,goat)were amplified by PCR and sequenced.The results showed that the sequence was highly conservative in bovine and caprine,respectively.Sensitivity test results showed that 0.2 ng DNA could be detected in the reaction system.The detection results of 5 entrusted animal-derived feeds showed that the method had great potential for the detection and identification of bovine and sheep(goat)derived ingredients in feed samples,and had important application value.Screening highly conserved single-copy sequences from mammals and birds as multi-species universal references and establishing quantitative detection methodQuantitative detection is an important method in assessing the adulteration of livestock and poultry products.Reliable and effective quantitative referencec sequence is an important prerequisite to solve this problem.Based on the exon data of 13 species from 11 families,nuclear DNA sequences with high homology(>95%)in mammals and birds were screened by means of comparative genomics and bioinformatics,and universal primers for multiple species were designed in sequence conservative regions.Tests in 18 animal genomic DNA showed that the sequence was highly universal and single-copy.Based on this multi-species quantitative reference,we further established and optimized a quantitative method for detection of animal-derived components.The relative error(R.E.)and relative standard deviation(R.S.D.)were both less than 25%.By introducing the concepts of genome equivalent(GE)and correction coefficient k,we realized the accurate quantitative detection of bovine,rabbit,dog,fox and mink components in mixed DNA samples.It provides reliable multi-species universal refference and detection methods for the accurate quantification of animal-derived components in livestock and poultry products.Establishment of multiplex quantitative detection method for animal-derived components in meat products based on multi-species universal referenceAiming at the "horse meat scandal" in Europe,combining the species-specific DNA sequence of bovine and equine,as well as multi-species universal quantitative reference,a multiplex qualitative and quantitative detection method for bovine(223 bp)and equine(197 bp)derived components was established.Aiming at the "false mutton incident" in China,combining the species-specific DNA sequence of sheep,fox and mouse,as well as multi-species universal quantitative reference,a multiplex qualitative and quantitative detection method for sheep and goat(237 bp),fox(211 bp)and muroidea(160 bp)derived components were established.All species-specific sequences were aligned with the reference genomes of 53 species by BLAST and ClustalW sequence alignment,and species-specific primers and probes were designed in the base difference region.The specificity of sequences,primers and probes was verified by conventional PCR and TaqMan fluorescent PCR in several species DNA,and these sequences were confirmed to be fixed copy number.The establishment of multiplex PCR system showed that there was no cross-interaction between primers and probes,which ensured simultaneous detection and quantification of multiple animal-derived components in a single system.Conservative experiments showed that the specific sequences and primers of each species were highly similar in their target species.Sensitivity test showed that the detection limit(LOD)was 0.05 ng DNA and the quantitative limit(LOQ)was 5%.The relationship between GE and Ct values was established by constructing the standard curves of species-specific sequence and internal reference sequence.Multiplex quantitative detection was carried out for mixed DNA samples and meat samples with different mass ratios.The results showed that R.E.and R.S.D.were less than 25%,which indicated that the established multiplex quantitative detection method had good detection accuracy and precision.In this study,species-specific nuclear DNA sequences were screened by comparative genome approach for provenance component detection,and conservative nuclear DNA sequences were screened as multi-species universal quantitative internal references.The qualitative and quantitative methods for detecting various provenance components were established.This provides the corresponding methods and candidate materials for provenance composition detection,and provides important technical support for food and feed safety. |
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