国内110株新生隐球菌临床株变种、基因型和交配型分析

Analysis of the varieties, genotypes and mating types of 110 clinical cryptococcal isolates from China

目的对国内部分地区的新生隐球菌（Cryptococcus neoformans）临床株进行分子流行病学调查，分析其变种、基因型和交配型的构成和分布。方法（1）PCR指纹分型法：以野生型噬菌体M13中针对小卫星DNA的核心,序列为单引物对模板进行PCR扩增，将所有受试菌株鉴定到8种主要基因型水平。（2）利用变种和交配型特异性引物扩增分型法，区分格鲁比变种（C．neoformansvar.grubii）、新生变种（C．neoformans var．neoformans）和格特变种（C．neoformans var．gattii），同时鉴定α和a交配型。结果110株临床株中，98株（89．1％）为格鲁比变种，均为VNⅠ基因型和α交配型；9株（8．2％）格特变种，包括VGⅠ基因型、α交配型8株（7．3％）和VGⅡ基因型、α交配型1株（0．9％）；2株（1．8％）为AD杂合体，VNⅢ基因型，-／α和α/-交配型各1株；1株（0．9％）为新生变种，VNⅣ基因型和a交配型。结论我国新生隐球菌临床株包含3个变种和AD杂合体。与国外情况比较，相似的是国内临床株中绝大部分为α交配型菌株，且格鲁比变种中的VNⅠ基因型占了其中的大部分；但未发现VNⅡ、VGⅢ和VGⅣ基因型菌株。

Objective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the constituents and distributions of the varieties, genotypes and mating types （MAT） of them. Methods （1） PCR fingerprinting and PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer. Genotypes of the 110 cryptococcal isolates from China were assigned by comparison with the reference strains of the 8 major molecular types loaded on gel. （2） Identification of the varieties and mating types was carried out by PCR using the specific primers of the varieties and mating types. Results Of the 110 chnical cryptococcal isolates, strains of Cryptococcus neoformans var. grubii with genetype VNⅠ and mating type MATα were the most representative ones （89.1%） followed by strains of C. neoformans var. gattii （8.2%） including isolates of genotype VGⅠ, mating type MATch（7.3% ） and genotype VGⅡ, mating type MATch（0.9% ） ; AD hybrids with the genotype VNⅢ, mating type MAT-/α and genotype VNⅢ, mating type MATα/- （ 1.8% ） ; and isolate of C. neoformans var. neoformans with the genotype VNⅣand mating type MATa （0.9%）. Conclusion Of the clinical isolates from China, all three varieties and AD hybrids are found. The vast majority （〉99% ） of strains possess the α allele in MAT locus and most of them are C. neoformans var. grubii with the genotype VN Ⅰ , which accord with the data of most studies of clinical molecular epidemiology in other geographic areas. However, no genotype of VN Ⅱ, VGⅢ and VGⅣ isolates are found in this study.