| Monitoring and detecting amantadine residues in chickens for safety purposes are difficult.It is inconvenient to monitor amantadine in actual applications since the target tissues with residues have not been reported.Among the researches on amantadine residue detection in China and many other countries,most used the chromatography-mass spectrometry technology for residue confirmation.However,the approach requires expensive equipment and specialized operating skills and the pretreatment process is complex,so it is not suitable for actual screening.Therefore,it is necessary to establish a more practical monitoring system and a new screening method to effectively monitor amantadine residues in chicken products.Searching for biomarkers based on transcriptomics provides a novel method of screening banned additives in animal products,and similar drugs could also be monitored effectively.By exploring the accumulation and elimination of amantadine residues in different tissues of broiler chickens,discussing the transcriptomic changes of amantadine in broiler chicken breast muscle and liver tissues and screening differentially expressed genes(DEGs)to identify candidate transcriptional biomarkers,this study established a monitoring method and an optimized model for monitoring amantadine residues in broiler chickens.This study aimed to provide a basis for target tissue selection of amantadine residues in broiler chicken products.The main contents and results of this work are as follows:(1)LC-MS/MS detection and elimination of amantadine residues in breast muscle,liver and plasma tissues of broiler chickens.The results showed that the samples were extracted and purified by the QuEChERS method.The LODs and LOQs of amantadine were 0.30 ?g/kg and 1.00 ?g/kg in breast muscles,0.50 ?g/kg and 1.67 ?g/kg in livers,and 0.34 ?g/kg and 1.10 ?g/kg in plasma.The recoveries were 88.5%-92.6% in breast muscles,88.5%-93.6% in livers,and 87.2%-95.5% in plasma,and their relative standard deviations were 4.0%-5.1%,4.4%-5.7% and 5.7%-6.3%,respectively,which were in line with the requirements of residual analysis.The residual concentrations of amantadine in chicken breast muscle,liver and plasma tissues rose with the increase of the dosage,and gradually decreased until eliminated as the days after drug withdrawal elapsed.The residual concentrations of amantadine in chicken breast muscle and plasma tissues were relatively close,the half-lives of elimination were 9.7h and 8.6h respectively,and the elimination rates were relatively fast.The residual concentrations of amantadine in liver tissues during the whole experimental period were higher than in breast muscles and plasma,the half-life of elimination was 11.1 h,and there was still a high residual concentration in liver tissues 312 hours after amantadine withdrawal.Therefore,it was slower for the amantadine residues in chicken liver tissues to disappear than in breast muscle and plasma tissues,and the chicken liver tissue could be used as a target tissue to detect illegal use of amantadine.(2)The transcriptomic changes of amantadine in breast muscle and liver tissues of broiler chickens.The results indicated that a total of 170 DEGs were screened from chicken breast muscle tissues after amantadine was fed.Among the genes,120 were up-and 50 were down-regulated.The gene ontology(GO)terms for these genes mainly existed in hydrolase activities,immune reactions and chemokine activities.The significantly enriched Kyoto Encyclopedia for Genes and Genomes(KEGG)pathways existed in phagosomes,cell adhesion molecules(CAMs),lysosomes,and extracellular matrix(ECM)receptors.From the chicken liver tissues,172 DEGs were screened,among which 116 were up-and 56 were down-regulated.The GO terms of these DEGs were related to the functions such as catalytic activities,metabolic activities,oxidation-reduction activities,immune reactions and cofactor binding.The significantly enriched KEGG pathways existed in the metabolism,CAM,ECM receptor reaction and drug metabolism-cytochrome P450.According to the fold-change(FC),significance level,functional annotation and possible biological process of DEGs,11 and 9 candidate DEGs related to amantadine treatment were further screened from chicken breast muscle and liver tissues,respectively.In addition,the quantitative real-time polymerase chain reaction(qRT-PCR)verification results showed exact concordance with the RNA-seq data.Principal components analysis(PCA)on the q RT-PCR data resulted in separation of treated samples from control samples in both tissues.The results provided a basis for identification of transcriptional biomarkers for detecting amantadine residues in breast muscle and liver tissues of broiler chickens.(3)The establishment and optimization of the amantadine monitoring method based on transcriptional biomarkers.The discriminant analysis(DA)results showed that 95.3% of the samples for which cross-validation was performed could be correctly classified when 20 target genes in the breast muscle and liver tissues were combined,which improved the discrimination accuracy by 10.9% and 3.1% for the two tissues,respectively.Therefore,with target genes in the two tissues in the process of searching for biomarkers,the samples could be distinguished more accurately and the number of false positives was reduced.In addition,11 genes were filtered based on the VIP value at the same time,and they were THRSP,DIO1,ETNPPL,GPAM,SIK1,ADRA1 D and RET of the liver tissue and ENSGALG00000000162,PDK4,PTGDS and CCL4 of the breast muscle tissue.The samples between the treatment group and control group could be distinguished better with this group of genes as biomarkers,and the correct classification ratio was above 95.3% for cross-validation of the DA results,which was completely consistent with the results of the 20 genes in the two tissues.The DA results of eight samples based on the 11 genes of the two tissues were completely correct,which verified the validity and correctness of this group of genes as biomarkers.Therefore,the group of 11 genes in breast muscle and liver tissues could serve as biomarkers to monitor amantadine abuse in chicken breeding. |
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