安徽省兔支气管败血波氏杆菌的分离鉴定及PCR诊断方法的建立

Isolation,Identification and Development of PCR Assay for Detection of Bordetella bronchiseptica Infection in Rabbits in Anhui Province

目的:从皖北多个大型兔场无菌采集发病兔肺脏进行兔支气管败血波氏杆菌(Bb)分离鉴定,并建立PCR诊断方法,为养兔场及时了解Bb的流行状况及本病的防控提供依据。方法:用5%绵羊血改良的B-G培养板对无菌采集的兔病变肺脏病料进行细菌培养,对分离菌株的形态、血清、生化特性等进行研究,并利用16S rRNA-PCR扩增技术加以验证;针对Bb表面特有的丝状血凝素(FHA)蛋白基因序列设计1对引物,建立检测Bb的PCR方法,并对反应条件进行优化。结果:本研究所得的Bb菌株在形态学、血清学、生化特性与标准Bb菌株一致,16S rRNA-PCR鉴定方法在1492 bp处扩增出目的条带,基因序列与Bb16S rRNA(E06073)同源性达到99.1%;新建的PCR方法25 mmol/L MgCl_(2)1.0μL、退火温度为58℃、30个循环),可扩增出特异醒目的目的条带(约730 bp);特异性试验显示该方法对兔巴氏杆菌、兔魏氏梭菌、金黄色葡萄球菌和兔大肠埃希氏菌检测均为阴性,敏感性试验显示该方法在Bb菌液浓度仅为7.2×10^(3)CFU/mL时仍能获得目的条带。结论:本研究从兔肺脏病料中分离的菌株经鉴定为Bb,并建立了检测该细菌的PCR方法,可用于临床鉴别和诊断兔支气管败血波氏杆菌病。

Objective:Isolation and identification of Bordetella bronchiseptica(Bb)from the lungs of infected rabbits collected from several large rabbit farms in the north of Anhui Province,and establishment of PCR diagnostic method,in order to understand the epidemic situation of Bb and provide the basis for the prevention and control of the disease.Methods:The B-G culture plate modified by 5%sheep blood was used to culture rabbit lung disease materials,the morphology,serology and biochemistry of the isolated strains were studied and validated by 16S rRNA-PCR.A pair of primers were designed for the gene sequence of the specific filamentous hemagglutinin(FHA)protein on the Bb surface,and a PCR method for the detection of Bb was established and the reaction conditions were optimized.Results:The morphological,serological and biochemical characteristics of Bb strains obtained in this study were consistent with those of standard Bb strains,the 16S rRNA-PCR identification method amplified the target band at 1492 bp,and the homology of the gene sequence to Bb 16S rRNA(E06073)was 99.1%.When the reaction was added with 1.0μL 25 mmol/L MgCl _(2),annealing temperature at 58℃and 30 cycles),the specific eye-catching bands(about 730 bp)could be amplified by the new PCR method;the specificity test showed that the method was negative for Pasteurella Multocida,Clostridium Perfringens,Staphylococcus aureus and Escherichia coli,the sensitivity test showed that the target band could be obtained when the concentration of Bb solution was only 7.2×10^(3) CFU/mL.Conclusion:In this study,the strain isolated from rabbit lung disease was identified as Bb,and a PCR method was established to detect the bacteria,it can be used for clinical differentiation and diagnosis of Bordetella bronchiseptica in rabbits.