以血培养为基础的布鲁菌快速鉴定方法研究

Study on rapid identification of Brucella based on blood culture

目的 探讨快速、简便的以血培养为基础的布鲁菌鉴定方法，为布鲁菌病的及时诊治提供依据。方法 应用培养仪进行血培养，通过观察阳性报警瓶生长曲线特征，对可疑布鲁菌生长的培养物通过革兰染色和生化反应进行快速鉴定。验证采用传统的阳性血清、单相特异性血清（A、M和R）凝集试验，布鲁菌噬菌体裂解试验（Tb、BK2）对布鲁菌可疑菌株进行种／型鉴定。对鉴定为布鲁菌的菌株采用PCR方法［布鲁菌表面蛋白-31（BCSP31）-PCR和牛种、羊种、绵羊附睾和猪种布鲁菌（AMOS）-PCR］鉴定分型。结果 61份培养物细菌生长曲线特征：迟缓期曲线较平坦，生长期曲线短，稳定期曲线平坦；在血培养仪上，72 h左右出现阳性报警。64株（其中3人培养2次）菌株尿素水解试验、革兰染色、柯氏染色鉴定为布鲁菌。选取27株菌株经细菌学鉴定为羊种布鲁菌，其中，21株为羊种Ⅲ型，6株为羊种Ⅰ型；经BCSP31-PCR鉴定均为布鲁菌，经AMOS-PCR方法鉴定均为羊种布鲁菌。结论 布鲁菌的快速鉴定可以缩短培养和鉴定时间，通过经典细菌学和PCR方法验证，此方法鉴定布鲁菌结果快速、可靠。

Objective To explore a rapid and simple method for identifying Brucella, and to provide further evidence for clinical diagnosis. Methods Bact/Alert blood culture system was used to culture whole blood samples, suspicious Brucella positive culture samples were screened by observing growth curve of the positive alarming bottles, and gram staining for blood smears and rapid biochemistry test was performed to identify Brucella initially. Traditional identification methods, including agglutination tests with positive serum and specific single phase serum （A, M and R） and Brucella bacteriophage lytic test, were used to identify suspected isolates to species or type. Clinical specimens from patients identified to be positive with Brucella were further identified and characterized by and PCR method [Brucella surface protein-31 （BSCP31）-PCR and Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis species-specific PCR （AMOS-PCR）]. Results The growing curve of the 61 clinical samples showed the following features, including flat lag growth phage, about 72 h of time to positivity, shorter logarithmic growth phase and flat stationary phase. The 64 isolates （3 of them were cultured for 2 times） were further identified to be Brucella with urea hydrolysis tests, gram staining and Kovac＇s staining. Twenty-seven strains were identified to be Brucella melitensis strains using bacteriological methods. Among the 27 of 64 strains, 21 of which were Brucella melitensis biovar 3 and 6 of them were Brucella melitensis biovar 1; the results of BCSP31-PCR test confirmed that the 27 Brucella strains were identified to be Brucella melitensis, the result of AMOS-PRC test again confirmed that 27 Brucella strains were Brucella melitensis. Conclusions The rapid Brucella identification method could shorten the identification time. Verified by conventional method and PCR method, the new method could rapidly and reliably identify Brucella strains.