Cerekci Ayşe, Kılıç Selçuk, Bayraktar Mehmet, Uyanık M Hamidullah, Yaşar Ekrem, Esen Berrin
Kahramanmaraş State Hospital, Microbiology Laboratory, Kahramanmaraş, Turkey.
Mikrobiyol Bul. 2011 Jul;45(3):392-400.
Brucellosis which is a worldwide zoonotic disease, still constitutes a major public health problem in rural areas of Turkey. The aim of the present study was to evaluate the species and biovar distribution of 187 presumptive Brucella strains isolated from patients inhabiting at the provinces in Eastern, South Eastern and Mediterranean regions over a 7-years period (from 2001 to 2007) and to compare multiplex real-time-polymerase chain reaction (M-RT-PCR) and conventional biotyping for the differentiation of three Brucella species. The isolates were identified at genus level by conventional microbiological methods and classified using the classical Brucella species biotyping scheme based on CO2 requirement for growth, urease activity, H2S production, sensitivity to basic fuchsin and thionin (20 and 40 µg/ml), lysis by Tbilisi and Berkeley phages, and agglutination with monospecific antisera for A and M antigens. All Brucella isolates were identified as Brucella melitensis biovar 3. M-RT-PCR assay targeted bcsp31 gene and the specific integration of IS711 elements within the genome of the respective Brucella species. For the identification of Brucella spp. The primers and probes which targeted the bcsp31 gene were used. The Brucella abortus primers and probe set targeted the specific insertion of an IS711 element downstream of the alkB gene, whereas the B.melitensis primers and probe set targeted the insertion of an IS711 element downstream of BMEI1162. M-RT-PCR results were found to be 100% compatible with the reference conventional typing methods. Due to its high sensitivity, the M-RT-PCR assay could be a valuable tool for the rapid detection and differentiation of Brucella species in clinical samples which usually have very low bacterial load. These findings indicated that B.melitensis biovar 3 was by far the most frequent species for human brucellosis in these specific regions of Turkey and multiplex-RT-PCR seemed to be promising in the detection and differentiation of Brucella species.
布鲁氏菌病是一种全球性人畜共患病，在土耳其农村地区仍然是一个主要的公共卫生问题。本研究的目的是评估在7年期间（2001年至2007年）从居住在东部、东南部和地中海地区省份的患者中分离出的187株疑似布鲁氏菌菌株的种类和生物变种分布，并比较多重实时聚合酶链反应（M-RT-PCR）和传统生物分型法对三种布鲁氏菌的鉴别能力。通过传统微生物学方法在属水平上鉴定分离株，并根据生长对二氧化碳的需求、脲酶活性、硫化氢产生、对碱性品红和硫堇（20和40μg/ml）的敏感性、第比利斯和伯克利噬菌体的裂解作用以及与A和M抗原单特异性抗血清的凝集反应，使用经典的布鲁氏菌种类生物分型方案进行分类。所有布鲁氏菌分离株均被鉴定为马尔他布鲁氏菌生物变种3。M-RT-PCR检测针对bcsp31基因以及各个布鲁氏菌物种基因组内IS711元件的特异性整合。为了鉴定布鲁氏菌属，使用靶向bcsp31基因的引物和探针。流产布鲁氏菌引物和探针组靶向alkB基因下游IS711元件的特异性插入，而马尔他布鲁氏菌引物和探针组靶向BMEI1162下游IS711元件的插入。发现M-RT-PCR结果与参考传统分型方法100%相符。由于其高灵敏度，M-RT-PCR检测可能是临床样本中布鲁氏菌物种快速检测和鉴别的有价值工具，临床样本中的细菌载量通常非常低。这些发现表明，在土耳其的这些特定地区，马尔他布鲁氏菌生物变种3是迄今为止人类布鲁氏菌病最常见的物种，多重RT-PCR在布鲁氏菌物种的检测和鉴别方面似乎很有前景。
