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首页> 外文期刊>Journal of Clinical Microbiology >Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.
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Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.

机译：使用嵌套引物基因扩增测定法快速，灵敏地检测麻风分枝杆菌。

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摘要

By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.
机译：通过使用一组四个嵌套的寡核苷酸引物，开发了一种用于检测和鉴定麻风分枝杆菌的两步聚合酶链反应测定法，该测定法不需要使用放射性标记的杂交探针。巢式引物程序从麻风分枝杆菌基因组DNA扩增了347个碱基对的产物。从其他19种分枝杆菌物种，19种非分枝杆菌物种，小鼠细胞或人类细胞的DNA中未产生扩增产物。观察到与另外三种分枝杆菌属，即“ M. lufu”，s。simiae和M. smegmatis的次要扩增产物。这些产物容易通过大小和限制酶切割模式与麻风分枝杆菌产物区分开。该测定法可从含有低至3 fg麻风分枝杆菌基因组DNA的样品中扩增347个碱基对的产物-单个细菌中的DNA量。该测定法还扩增了从感染动物和人的组织活检标本中分离出的麻风杆菌的粗裂解物中的靶序列。从样品制备到数据分析的整个测定过程都可以在8小时内完成。

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