| 重庆地区兔腹泻病原菌分离鉴定及检测方法的建立 |
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| 引用本文：许国洋，沈克飞，徐登峰，付利芝，张素辉，王孝友，杨 柳.重庆地区兔腹泻病原菌分离鉴定及检测方法的建立[J].西北农业学报,2018,27(8):1097~1103 |
| DOI:10.7606/j.issn.1004-1389.2018.08.003 |
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| 基金项目:重庆市农业发展基金（16408）；重庆市农业技术推广与服务补助资金（14461）。 |
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| 中文摘要:对重庆地区兔细菌性腹泻病原菌进行分离鉴定并建立相应检测方法，为该病的检测及防控奠定基础。通过临床诊断与细菌分离培养，对引起兔腹泻的病原菌进行分离，利用细菌通用引物扩增分离菌16S rDNA基因序列，测序分析后构建系统进化发育树。同时，依据各病原菌特异性序列设计引物，建立PCR检测方法。结果表明，共分离到4种病原菌，分子生物学鉴定为：铜绿假单胞菌、产气荚膜梭菌、金黄色葡萄球菌和大肠杆菌；依据铜绿假单胞菌外毒素eta基因、产气荚膜梭菌a毒素基因、金黄色葡萄球菌耐热核酸酶nuc基因和大肠杆菌外膜蛋白eae基因设计特异性引物，成功建立多重PCR检测方法，可从4种病原菌基因组混合物中扩增出大小分别为1 043、324、200和609 bp的目的条带，目的菌株最低检出浓度为10 cfu·mL-1。对重庆地区200 份临床样本进行检测，发现4种病原菌普遍存在，其中，金黄色葡萄球菌单独感染检出率高达37.6%，与大肠杆菌的混合感染检出率达28.0%。 |
| 中文关键词:兔腹泻 病原菌 分离鉴定 多重PCR 混合感染，检出率 |
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| Isolation and Identification of Pathogenic Bacteria of Rabbit Diarrhea in Chongqing and Establishment of Its Detection Method |
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| Abstract:The purpose of this study is to isolate and identify the pathogenic bacteria of rabbit diarrhea in Chongqing and establish its detection method. The pathogenic bacteria was isolated by the method of clinical diagnosis and bacterium separation technology. The 16S rDNA gene, which was augmented by universal primer for bacteria and used to build the phylogenetic tree, was carried out in the NCBI Blast comparison. And PCR detection method was established based on the specific primers of the bacterial strains. The molecular identification result showed that Pseudomonas aeruginosa, Clostridium perfringens, Staphylococcus aureus and Escherichia coli were successfully separated based on the phylogenetic analysis. The multiple PCR method which was established according to the gene of eta,toxin a,nuc and eae, could amply the purpose strip with a fragment of 1 043 bp, 324 bp, 200 bp and 609 bp.The minimum detectable concentration of the pathogenic bacteria could be checked out with the concentration of 10 cfu·mL-1.The 200 clinical samples test results showed that the four kinds of pathogenic bacteria were ubiquitous. And Staphylococcus aureus was independently checked out with the highest detection rate of 37.6%. In addition, the mixed infection of Staphylococcus aureus and Escherichia coli was discovered with the detection rate of 28.0% by the method of multiple PCR. Four kinds of pathogenic bacteria of rabbit diarrhea were isolated based on the research. And the multiple PCR method which had been successfully established laid foundation for detection and prevention of rabbit bacterial diarrhea. |
| keywords:Rabbit diarrhea Pathogenic bacteria Isolation and identification Multiple PCR Mixed infection Detection rate |
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