Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades
Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades
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DOI：
10.1128/jcm.01496-07
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发表时间：
2008-01-01
影响因子：
9.4
通讯作者：
Keim, Paul
中科院分区：
文献类型：
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作者：
Foster, Jeffrey T.;Okinaka, Richard T.;Keim, Paul
布鲁氏菌属的成员在世界范围内被认为是野生动物和牲畜的病原体，并且是人类最常见的人畜共患感染生物。一般而言，布鲁氏菌在动物中表现出一系列宿主特异性，导致至少鉴定出7种布鲁氏菌。各种布鲁氏菌属物种的基因组高度保守，这使得物种的区分极具挑战性。然而，我们在管家基因和其他基因中发现了单核苷酸多态性（SNP），这些基因区分了7个主要的布鲁氏菌物种或分支，从而使我们能够开发实时PCR。基于这些SNP的分析。用这些试验筛选了338个不同分离株的不同组，正确鉴定了每个分离株与其先前确定的布鲁氏菌分支。七个分支特异性检测的DNA浓度小于10 fg，表明高水平的灵敏度。这种基于SNP的方法将样本置于系统发育框架中，从而可以在克隆细菌的谱系中进行可靠的比较，并为基因分型提供坚实的基础。这些PCR检测提供了一种快速和高度敏感的方法区分布鲁氏菌的主要群体，这将是有价值的临床和法医应用。
Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR. assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.