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首页> 外文期刊>Journal of Clinical Microbiology >High-Throughput Method for Detecting Genomic-Deletion Polymorphisms
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High-Throughput Method for Detecting Genomic-Deletion Polymorphisms

机译：检测基因组缺失多态性的高通量方法

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DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.
机译：DNA芯片已成功用于包括结核分枝杆菌在内的不同微生物，可检测相对于参考菌株的基因组缺失。然而，微阵列系统的成本和复杂性是其在大规模研究中广泛使用的障碍。为了评估细菌种群中大序列多态性（LSP）或插入缺失事件的程度和作用，我们开发了一种称为“ deligotyping”的技术，该技术可将多重PCR产物与膜结合的高特异性寡核苷酸探针杂交。该方法具有成本低廉的优点，并且能够针对43个基因组区域同时询问40多个细菌菌株。膜上代表的缺失选自先前的比较基因组研究和正在进行的微阵列实验。设计用于这些缺失的高度特异性的探针，并将其附着在膜上，以与菌株衍生的靶标杂交。通过多重PCR产生靶标，允许在单个反应中同时扩增43个不同的基因组基因座。为了验证我们的方法，分析了使用高密度微阵列分析的100个菌株。膜以99.9％的灵敏度和98.0％的特异性准确检测通过微阵列方法鉴定的缺失。地物分型技术可以快速可靠地筛查大量 M。结核病分离株。该技术可用于深入了解 M的流行病学，基因组进化和种群结构。结核，并且可以用于研究其他生物。

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