Plikaytis B B, Gelber R H, Shinnick T M
Hansen Disease Laboratory, Centers for Disease Control, Atlanta, Georgia 30333.
J Clin Microbiol. 1990 Sep;28(9):1913-7. doi: 10.1128/jcm.28.9.1913-1917.1990.
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.
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，该方法无需使用放射性标记的杂交探针。嵌套引物程序从麻风分枝杆菌基因组DNA中扩增出一个347个碱基对的产物。来自其他19种分枝杆菌、19种非分枝杆菌、小鼠细胞或人类细胞的DNA均未产生扩增产物。在另外三种分枝杆菌，即“鲁氏分枝杆菌”、猴分枝杆菌和耻垢分枝杆菌中观察到少量扩增产物。这些产物通过大小和限制性内切酶切割模式很容易与麻风分枝杆菌产物区分开来。该测定法可以从含有低至3 fg麻风分枝杆菌基因组DNA的样品中扩增出347个碱基对的产物，这是单个杆菌中的DNA量。该测定法还扩增了从感染动物和人类的组织活检标本中分离出的麻风分枝杆菌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可以在不到8小时内完成。
