摘要目的 建立一种核糖体基因内间隔区(IGS)的特异性PCR扩增和序列分析方法,用于快速鉴定格特隐球菌VGⅡ基因型及其亚型.方法 选取新型和格特隐球菌核糖体IGS区中可变度最高的1区为靶点,经ClustalX 2多重比对后,设计特异性扩增格特隐球菌VGⅡ基因型的引物用于PCR分析.通过扩增新型和格特隐球菌其余基因型以及其他致病酵母菌来评价引物的特异性,并对阳性扩增片段(VGⅡ基因型)进行测序和序列分型研究.结果 基于核糖体IGS区的特异PCR引物扩增所有受试格特隐球菌VGⅡ基因型菌株均为阳性,而新型和格特隐球菌其余基因型以及其他致病酵母菌均为阴性.序列分型显示,在扩增片段的72、79和104 bp处存在3个多态性位点,可用于区分VGⅡ基因型中的不同亚型.结论 该研究建立的特异性PCR扩增方法可快速、准确地鉴定格特隐球菌VGⅡ基因型,对扩增片段的序列分型可初步筛查高致病性的VGⅡa亚型.

abstractsObjective To establish a PCR method for rapid identification and sequence typing of VG Ⅱ allele of the intergenic spacer region (IGS) of Cryptococcus gattii.Methods Since IGS1 was of high sequence variation,multiple alignments were conducted by ClustalX 2 in IGS1 of Cryptococcus gattii and Cryptococcus neoformans,and then primer sets specific to genotype VG Ⅱ was designed for PCR analysis.The specificity of the primer pair was detected by amplification of the other genotypes in Cryptococcus gattii,Cryptococcus neoformans,and other pathogenic yeasts.The amplified fragments from VG Ⅱ genotype were sequenced and subtyped.Results Using the PCR analysis developed in this study,all VG Ⅱ genotype strains tested were amplified,whereas no amplification was obtained from other genotypes or yeast species involved herein.Three polymorphic nucleotide sites at 72,79 and 104 bp in the fragment amplified could be used to distinguish sub-genotypes within VG Ⅱ genotype.Conclusions The PCR analysis developed in this study can be used for rapid identification of genotype VG Ⅱ of Cryptococcus gattii.The sequence typing based on the amplified fragment from IGS1 may be performed for screening the highly virulent sub-genotype VGⅡ a.