Targeted Sequencing And Metagenomic Sequencing Were Used To Compare The Distribution Characteristics Of Drug Resistance Genes In Cattle Farms

For microbial communities between humans,animals,the environment,and mixed niches among the three,the selection pressure caused by heavy antibiotic use confers a survival advantage to bacteria with resistance genes(ARGs),that is,bacterial resistance sex.The drug resistance of animal-derived bacteria mainly comes from the irregular use of a large number of antibiotics in animal husbandry,which not only seriously harms the development of animal husbandry,but also poses a serious threat to public health security.As a new type of pollutant,ARGs carried by drug-resistant strains will not disappear due to the demise of bacteria.Once they enter the environment,they will persist for a long time and spread in the flora through the mediation of bacteriophages and plasmids,polluting soil and water bodies.,and ultimately harm human health along the food chain.The detection and research methods of ARGs in the environment are common PCR and fluorescent PCR.With the development of high-throughput sequencing technology,metagenomic sequencing is widely used,especially for the detection of ARGs in complex environmental samples such as farms.technical means,but this method is difficult to detect specific genes at low levels.As a rapid and economical method to detect known and novel variant genes in specific gene sets or genomic regions,targeted sequencing has been widely used in the diagnosis of genetic diseases and tumor companion diagnosis,but it is rarely used in the detection of ARGs.In order to understand and master the distribution characteristics of ARGs in the environment of some dairy,beef and breeding cattle farms in Jilin Province,this study used the Ampli Seq Designer design software provided by Thermo Fisher to select representative 31 different antibiotics from the CARD database 716 pairs of amplicon primers were designed and synthesized for 364 ARGs of the category,and the Ion Torrent sequencing platform was used to conduct targeted sequencing of environmental samples from different dairy,beef and breeding cattle farms,and combined with metagenomic sequencing to compare the two sequencing methods difference.The results of the two sequencing methods showed that in the environmental samples of each farm,the dairy farms had the most types and the most abundant ARGs,followed by the beef cattle farms.There are differences in the types of ARGs obtained.In the targeted sequencing results,69 kinds of ARGs were detected in each sample,and there were 34 kinds of ARGs with a sequencing depth greater than 10 times;in the metagenomic sequencing results,50 kinds of ARGs were detected in each sample,and the sequencing depth Only tet W,rrs D,EF-Tu,and rrs B were shared ARGs more than 10 times larger.The comparison of the two sequencing results showed that there were significant differences in the content of the same ARGs in the same sample.In order to verify which sequencing method can better reflect the content of drug resistance genes,based on the sequencing results and combined with the strains containing Erm B,tet O,lnu C,APH(6)-Id,dfr A17 and other drug resistance genes preserved in our laboratory,a fluorescence quantitative method was established.PCR method,validated for sensitivity,specificity,and reproducibility,for quantitative analysis of sequenced samples.The quantitative detection of Erm B,tet O,lnu C,APH(6)-Id and dfr A17 was closer to the results of targeted sequencing.The development of this study not only provides a useful attempt for the detection of drug resistance genes by targeted sequencing in complex environments,but also provides evidence for understanding and mastering the distribution characteristics of drug resistance genes in some dairy,beef and breeding cattle farms in Jilin Province.