Study On Transcriptive Biomarlers To Monitor β₂-agonists In Goat Muscle

The abuse of β₂-agonists in animal husbandry is a major topic at home and abroad. Thoughβ₂-agonists were controlled by detecting substance residues in different matrices, the application of newly designed and escaped drugs represents big problems. Therefore, developing effective test methods are important. β₂-agonists have the same mechanism, analysis of physiological changes caused by β₂-agonists on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. In this paper, for finding transcriptive biomarkers ofβ₂-agonists, through initial establishment of screening model, method validation, model optimization and sample verification, long-term, stable and efficient biomarkers were found and established as a monitoring model to provide a reference for the effective monitoring of β₂-agonists. The main contents and results are as follows:（1） The method based on targeted transcriptome approach was established. Results showed that biomarkers through literatures can make a distinction between treatment group and control, and also seperate treatment samples in different residue levels. QRT-PCR data demonstrated that ractopamine can influence gene expression levels of selected nine candidate genes, principal component analysis,cluster analysis and discriminant analysis results showed that the group of genes can be biomarkers to reflect the illegal use of β₂-agonists. After correlation analysis of the residues and qRT-PCR data of the treated animals, linear regression analysis indicated an association between the predictive value and the actual value, which reflected that changes of nine gene expression levels were specific to the treatment of ractopamine. Statistical analysis displayed that these genes are also suitable for describing different residue levels separately.（2） Combined with non-targeted tool for method optimization, there was a total of seventeen genes together with eight genes choosed by RNA sequence. PCA, HCA and DA results demonstrated that compared with the literature-based data, results of seventeen genes was improved, which displayed that a combination of targeted and untargeted approach was an ideal way for finding potential gene expression biomarkers. Further optimizing method by VIP value, six genes were selected from seventeen genes. Statistical analysis demonstrated that results of six genes was consistent with seventeen genes. Meanwhile, the selected six genes model facilitated the reduction of operation time and amount during actual operation, which will play a significant role in practical application in the future.（3） Method application and validation. Results showed that the selected six genes determined correctly of the twenty-five samples whether animals was treated with β₂-agonists or not, which further proved the validity and effectiveness of the method. In addition, only ractopamine was applied for treatment of animals in the experiment, but during the validation, another β₂-agonist mapenterol was detected, indicating that transcriptive biomarkers was sufficient and effective to monitor all theβ₂-agonists. With routine monitoring method facing challenges, such as ractopamine possess a discontinuation in a week, escape drugs and any other new drug of β₂-agonists, transcriptive biomarkers show great significance in monitoring β₂-agonists abuse in animal husbandry.