DNA芯片鉴定念珠菌种和氟康唑耐药基因ERG11突变

Application of DNA microarray in the identification of Candida spp. and mutations of ERGll gene restflting in fluconazole resistance

目的建立DNA芯片技术鉴定念珠菌种和氟康唑耐药基因ERG11突变。方法根据6种常见念珠菌内转录间隔（ITS2）区种特异性序列和白念珠菌ERG11基因中已证实可导致对氟康唑耐药的6种突变序列设计探针，制备DNA芯片，鉴定12条50bp的念珠菌种特异性序列和ERG11突变序列及34株念珠菌（其中白念珠菌29株，热带念珠菌、光滑念珠菌、都柏林念珠菌、近平滑念珠菌和克柔念珠菌各1株）。结果①芯片正确鉴定12条人工合成序列；②正确鉴定34株试验菌株的菌种；③正确鉴定29株白念珠菌ERG11基因中可致耐药的已知突变。敏感性和特异性均为100％：结论用DNA芯片进行念珠菌菌种鉴定和白念珠菌ERG11突变筛查，结果可靠。

Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERGll mutations resulting in fluconazole-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 （ITS2） region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.e., T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-four Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata, 1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGll gene. Sequencing was performed to analyze the sequence of ERGll in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the mutations were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krnsei. Both the sensitivity and the specificity of the microarray were 100%. Conclusion DNA microarray is a quite reliable method to identify Candida spp. and fiuconazole resistance-associated mutations in the ERG11 gene of C. albicans.