Lasker Brent A, Ran Yuping
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
J Clin Microbiol. 2004 Apr;42(4):1483-90. doi: 10.1128/JCM.42.4.1483-1490.2004.
Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of P. marneffei with a high discriminatory power (D = 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of P. marneffei.
马尔尼菲青霉是一种新出现的机会性双相真菌病原体，在东南亚地区呈地方性流行。研究了一种基于微卫星位点大小多态性分析的分型方法。从GenBank数据库中鉴定出三个含有微卫星的位点。设计了位于微卫星重复序列两侧的PCR引物，其中一组引物中的一个进行了荧光标记。然后通过自动毛细管电泳对PCR产物进行大小测定。正如对单倍体真菌所预期的那样，所有分离株的每个微卫星位点均观察到一条单带。多态性微卫星标记（PMM）分析检测到35株马尔尼菲青霉共有22种不同的等位基因类型，具有较高的鉴别力（D = 0.956）。微卫星I、II和III分别检测到14、10和7个等位基因。通过使用来自同一分离株的不同DNA制剂或对同一DNA制剂进行重复检测，证实了长度多态性的可重复性。在体外传代7至8周的8株分离株的PMM图谱与原始培养物相同，表明具有短期稳定性和可重复性。在其他双相真菌或人类DNA中未观察到PCR产物。对来自中国和泰国的分离株的等位基因频率进行比较，发现了不同的等位基因组合，这表明种群可能存在地理隔离。由于具有较高的鉴别力、可重复性和高通量潜力，PMM分析可能为马尔尼菲青霉的流行病学和监测调查提供一种良好的分型方法。
