Kurt Kevin, Alderborn Anders, Nilsson Mats, Strommenger Birgit, Witte Wolfgang, Nübel Ulrich
Robert-Koch-Institut, Burgstr. 37, 38855 Wernigerode, Germany.
J Clin Microbiol. 2009 Mar;47(3):577-85. doi: 10.1128/JCM.01347-08. Epub 2009 Jan 21.
We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analyzed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in the mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin-resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i.e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation with clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA based on a single-tube reaction. With a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typeability and 100% accuracy were achieved. The assay described here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable for the quick and cost-effective probing of large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome resequencing projects.
我们开发并测试了一种基于连接酶的检测方法，用于同时探究金黄色葡萄球菌分离株的核心基因组多样性和耐甲氧西林决定因素的分型。该检测方法使用寡核苷酸锁式探针，当它们与匹配的靶DNA杂交时，其两端通过连接反应连接在一起。随后，环化探针通过通用引物进行PCR扩增，并使用配备通用标签探针的微阵列进行分析。我们的锁式探针组包括靶向耐甲氧西林金黄色葡萄球菌（MRSA）中SCCmec盒的mecA、ccrB和ccrC基因诊断区域的寡核苷酸。这些探针可确定SCCmec盒的存在和类型（即SCCmec I至VI型）。另外的寡核苷酸用于询问从多位点序列分型（MLST）数据库中检索到的一些信息丰富的单核苷酸多态性。后一种探针能够探索分离株与MLST揭示的MRSA克隆谱系的系统发育关系。所描述的检测方法能够基于单管反应对MRSA进行多重基因分型。对于一组MRSA临床分离株和甲氧西林敏感金黄色葡萄球菌（n = 66），实现了100%的分型能力和100%的准确性。本文所述的检测方法提供了有价值的基因型信息，可有效补充现有的基因分型程序。此外，通过加入额外的锁式探针，该检测方法易于扩展，对于快速且经济高效地探究不同基因组位置的大量多态性（如通过当前正在进行的突变发现和基因组重测序项目确定的多态性）具有重要价值。
