DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
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DOI：
10.3791/3104
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发表时间：
2011-07-01
影响因子：
1.2
通讯作者：
Vissa, Varalakshmi
中科院分区：
文献类型：
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作者：
Jensen, Ronald W.;Rivest, Jason;Vissa, Varalakshmi
麻风传播的研究特别困难，因为麻风分枝杆菌不能在实验室培养。这种细菌的唯一来源是麻风病患者，并通过实验感染了螳螂和裸鼠。因此，现代流行病学中使用的许多方法不适用于麻风病的研究。尽管世界卫生组织1实施了广泛的全球麻风病药物治疗计划，但麻风病在许多国家仍然流行，每年约有25万新病例。(2)已绘制了麻风杆菌的整个基因组图谱(3，4)，并已确定了许多具有2个或更多碱基对的重复片段的基因座(称为微卫星和小卫星)。(5)麻风杆菌临床菌株在这些基因座上的串联重复片段(短串联重复序列，STR)的数量可能有所不同。(5，6，7)可变数目串联重复序列(VNTR)(5)分析用于区分不同菌株的麻风杆菌。有些基因座似乎比其他基因座更稳定，重复次数的变化较小，而另一些基因座的变化似乎更快，有时是在同一个患者身上。虽然某些VNTRs的变异性引发了关于它们是否适合菌株分型的问题(7，8，9)，但新出现的数据表明，分析稳定性不同的多个基因座可以作为一种有价值的流行病学工具。多基因座遗传变异分析(MLVA)(10)已被用于研究几个国家的麻风进化和传播，包括中国(11，12)、马拉维(8)、菲律宾(10，13)和巴西(14)。MLVA涉及多个步骤。首先，从临床活检或皮肤涂片(SSS)中提取细菌DNA和宿主组织DNA。(10)然后通过聚合酶链式反应(PCR)从提取的DNA中扩增出所需的基因座。每个反应使用4-5个不同基因座的荧光标记引物，在总共4个反应中扩增出18个基因座。(10)可对PCR产物进行琼脂糖凝胶电泳以验证所需DNA片段的存在，然后提交毛细管电泳法进行荧光片段长度分析(FLA)。以每一基因座重复拷贝数为已知数量的蚯蚓传代细菌的DNA作为阳性对照。然后使用峰值扫描仪软件检查FLA色谱图，并将片段长度转换为VNTR拷贝数(等位基因)。最后，对VNTR单倍型进行了模式分析，当与患者临床数据结合时，可以用来跟踪菌株类型的分布。
The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO1, leprosy remains endemic in many countries with approximately 250,000 new cases each year. (2) The entire M. leprae genome has been mapped(3,4) and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). (5) Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. (5,6,7) Variable number tandem repeat (VNTR) (5) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing(7,8,9), the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) (10) has been used to study leprosy evolution and transmission in several countries including China(11,12), Malawi(8), the Philippines(10,13), and Brazil(14). MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). (10) The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. (10) The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.