新生隐球菌格鲁比变种、新生变种和格特隐球菌的多重聚合酶链反应鉴定  

Identification of Cryptococcus neoformans vat. grubii, vat. neoformans and Cryptococcus gattii by multiplex PCR FENG

作　　者：冯晓博[1] 凌波[1] 付小花[2] 王磊[2] 任大明[3] 姚志荣[1] 

机构地区：[1]上海交通大学医学院附属新华医院皮肤科,200092 [2]同济大学环境科学与工程学院 [3]复旦大学遗传所遗传工程国家重点实验室

出　　处：《中华皮肤科杂志》2012年第12期870-873,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（30870108,31000549）

摘　　要：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Oligo 6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GPAlA、CLA4D和SODlgattii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GPAlA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。Objective To establish a multiplex PCR targeting the intergenic spacer regions （IGS） for the identification of ＂Cryptococcus neoformans vat. grubff, var. neoformans and Cryptococcus gattii. Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of 1GS1 region, which shows high sequence variability in the genome of Cryptococcus neo^ormans and Cryptococcus gattii., for the multiplex PCR. Then, the developed multiplex PCR was performed to identify 51 Cryptococcus neoormans 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 vat. gmbii, 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. gmbii strain and one vat. neo^ormans 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 vat. grubii, vat. neoformans, AD hybrid, and Cryptococcus gattii, with superior performance in comparison with common PCR and CGB medium-based culture.

关 键 词：隐球菌属 聚合酶链反应 基因型 

分 类 号：R446.6[医药卫生—诊断学]