CAJ | 학술논문

目的建立一种基于核糖体基因内间隔区(IGS)的多重聚合酶链反应(PCR),用于快速鉴定新生隐球菌新生变种、格鲁比变种和格特隐球菌.方法选取新生隐球菌和格特隐球菌IGS中变异度最高的Ⅰ区(IGSl)为靶点,经ClustalW2多重比对,并结合Olig06软件在不同序列位点设计针对新生隐球菌新生变种、格鲁比变种和格特隐球菌的引物用于多重PCR分析.通过51株新生隐球菌(VNⅠ-VNⅣ和VNB基因型)和41株格特隐球菌(VGⅠ-VGⅣ基因型)对该方法进行验证,并将该方法与已报道的CGB显色培养和采用特异性引物GPA1A、CLA4D和SOD1 gattii扩增格鲁比变种、新生变种和格特隐球菌的单一引物PCR方法进行比较.结果基于IGS的多重PCR分析成功鉴定所有92株新生隐球菌和格特隐球菌,对其他常见致病酵母的扩增均阴性,显示所设计引物较高的特异性；已报道的基于GPA1A和CLA4D引物PCR分别在鉴定2株和1株格特隐球菌时出现假阳性结果；CGB培养基在鉴定1株格鲁比变种和1株新生变种时出现假阳性结果.上述方法在鉴定时均未出现假阴性结果.结论建立的多重PCR可快速准确地鉴定新生隐球菌新生变种、格鲁比变种、AD杂合子和格特隐球菌,且优于已报道的单一引物PCR或CGB显色培养法.
목적 건립일충기우핵당체기인내간격구(IGS)적다중취합매련반응(PCR),용우쾌속감정신생은구균신생변충、격로비변충화격특은구균.방법 선취신생은구균화격특은구균IGS중변이도최고적Ⅰ구(IGSl)위파점,경ClustalW2다중비대,병결합Olig06연건재불동서렬위점설계침대신생은구균신생변충、격로비변충화격특은구균적인물용우다중PCR분석.통과51주신생은구균(VNⅠ-VNⅣ화VNB기인형)화41주격특은구균(VGⅠ-VGⅣ기인형)대해방법진행험증,병장해방법여이보도적CGB현색배양화채용특이성인물GPA1A、CLA4D화SOD1 gattii확증격로비변충、신생변충화격특은구균적단일인물PCR방법진행비교.결과 기우IGS적다중PCR분석성공감정소유92주신생은구균화격특은구균,대기타상견치병효모적확증균음성,현시소설계인물교고적특이성；이보도적기우GPA1A화CLA4D인물PCR분별재감정2주화1주격특은구균시출현가양성결과；CGB배양기재감정1주격로비변충화1주신생변충시출현가양성결과.상술방법재감정시균미출현가음성결과.결론 건립적다중PCR가쾌속준학지감정신생은구균신생변충、격로비변충、AD잡합자화격특은구균,차우우이보도적단일인물PCR혹CGB현색배양법.
Objective To establish a multiplex PCR targeting the intergenic spacer regions (IGS) for the identification of Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii.Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of IGS1 region,which shows high sequence variability in the genome of Cryptococcus neoformans and Cryptococcus gattii.,for the multiplex PCR.Then,the developed multiplex PCR was performed to identify 51 Cryptococcus neoformans strains representing genotypes VNI-VNIV and VNB as well as 41 Cryptococcus gattii strains representing genotypes VGI-VGIV.The identification results were compared with those from common PCR by using primers GPA1A,CLA4D and SODlgattii specific to Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii,respectively,as well as with those from the canavanine-glycine-bromothymol blue (CGB) medium-based culture.Results The developed multiplex PCR successfully identified the 92 Cryptococcus neoformans and Cryptococcus gattii.strains,and yielded negative results from the other tested pathogenic yeasts,which revealed a high specificity of the designed primers.False positive results were observed in the identification of two Cryptococcus gattii strains with GPA1A primer-based PCR,one Cryptococcus gattii strain with CLA4D primer-based PCR,one var.grubii strain and one var.neoformans strain with CGB culture,while no false negative results were observed in the detection of these Cryptococcus strains by any of these methods.Conclusions The developed multiplex PCR in this study can rapidly and accurately identify Cryptococcus neoformans var.grubii,var.neoformans,AD hybrid,and Cryptococcus gattii,with superior performance in comparison with common PCR and CGB medium-based culture.