CAJ | 학술논문

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.
목적 건립일충핵당체기인내간격구(IGS)적특이성PCR확증화서렬분석방법,용우쾌속감정격특은구균VGⅡ기인형급기아형.방법 선취신형화격특은구균핵당체IGS구중가변도최고적1구위파점,경ClustalX 2다중비대후,설계특이성확증격특은구균VGⅡ기인형적인물용우PCR분석.통과확증신형화격특은구균기여기인형이급기타치병효모균래평개인물적특이성,병대양성확증편단(VGⅡ기인형)진행측서화서렬분형연구.결과 기우핵당체IGS구적특이PCR인물확증소유수시격특은구균VGⅡ기인형균주균위양성,이신형화격특은구균기여기인형이급기타치병효모균균위음성.서렬분형현시,재확증편단적72、79화104 bp처존재3개다태성위점,가용우구분VGⅡ기인형중적불동아형.결론 해연구건립적특이성PCR확증방법가쾌속、준학지감정격특은구균VGⅡ기인형,대확증편단적서렬분형가초보사사고치병성적VGⅡa아형.
Objective 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.