山羊奇异变形杆菌分离鉴定及其16S-23SrRNAISR序列RFLP分析

Isolation and Identification of Proteus mirabilis from Goat and the Analysis of Its 16S-23S rRNA ISR Sequence by RFLP

2012年初,山东菏泽某羊场的羊群发病,从发病羊器官分离到2株病原细菌。对病原细菌进行鉴定,并对其与已知同种异源菌株相似性差异进行分析。从患病山羊内脏器官分离细菌,经形态特征、培养特性、生化试验、血清学试验及致病性试验进行鉴定;再通过设计通用引物扩增16S-23SrRNA ISR(intergenic spacer region)序列,将PCR产物经HinfⅠ单酶切获得3条可视条带,同时对扩增条带中的主带测序并进行系统发育分析。结果表明,分离菌株为奇异变形杆菌;分离菌株同本实验室保存的兔源与鸡源奇异变形杆菌PCR-RFLP结果一致;分离菌株PCR产物同GenBank收录的HI4320株奇异变形杆菌及本实验室保存的兔源与鸡源奇异变形杆菌进行序列比较,分离羊源菌株与兔源菌株相似性为94.8%、与鸡源菌株相似性为96.0%～98.2%,与人源HI4320株相似性为96.9%。研究证实发病羊致病病原为奇异变形杆菌,其与鸡源、兔源和人源奇异变形杆菌的亲缘关系较近。

At the beginning of 2012, a disease occurred in a goat farm in Heze City and two strains of pathogen were isolated from the infected goats. In order to identify the infected bacteria and analyze the homology between isolated strains and heterologous strains, bacteria were isolated from infected goats internal organs and were identified by morphologic characteristics, cultural characteristic, biochemistry test, serologic test and pathogenicity test; A pair of universal prim- ers was designed to amplify 16S-23S rRNA ISR （intergenic spacer region） gene^and three visible straps were observed when PCR products were cut by Hinf I , at the same time the main strap of PCR straps was sequenced and analyzed by phyletic evolution. The results showed that isolated strains were Proteus mirabilis; the result of PCR-RFLP of isolated strains and Proteus mirabilis from rabbit and chicken was the same; The homology was 94.8% between isolated strains from goat and Proteus mirabilis from rabbit, 96.0%-98.2% between isolated strains from goat and Proteus mirabilis from chicken and 96.9 0/60 between isolated strains from goat and Proteus mira- bilis from human. Therefore primary bacterium of the case was Proteus mirabilis the genetic re- lationship of isolated strains with Proteus mirabilis from rabbit, chicken and human was closer.