中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (2): 108-111.

• 论著 • 上一篇    下一篇

DNA芯片检测泌尿生殖道病原体及耐药的研究

周文明1, 赵建龙2, 杨森1, 曹慧敏2, 李伟1, 沈玉君1, 张书梅1, 杜文辉1, 张学军1   

  1. 1. 安徽医科大学皮肤性病研究所、第一附属医院皮肤性病科 合肥 230022;
    2. 中国科学院上海微系统与信息技术研究所
  • 收稿日期:2004-02-15 出版日期:2005-02-15 发布日期:2005-02-15
  • 通讯作者: 张学军,E-mail:ayzxj@mail.hf.ah.cn E-mail:ayzxj@mail.hf.ah.cn

Detection of Pathogens of Urogenital Infections and Their Drug-resistant Types by a DNA Chip

ZHOU Wen-ming 1, ZHAO Jian-long2, YANG Sen1, CAO Hui-min2, LI Wei1, SHEN Yu-jun1, ZHANG Shu-mei1, DU Wen-hui1, ZHANG Xue-jun1   

  1. Department of Dermatology and Venereology, First Affiliated Hospital, Institute of Dermatology, Anhui Medical University, Hefei 230022, China
  • Received:2004-02-15 Online:2005-02-15 Published:2005-02-15

PDF

202

赞

摘要： 目的 研制一种DNA芯片,结合多重PCR方法快速检测泌尿生殖道炎症3种病原体及其耐药类型。方法 根据泌尿生殖道病原体淋球菌、沙眼衣原体和解脲脲原体的基因保守序列设计引物和探针,分别检测3种病原体;根据淋球菌gyrA、parC和16srRNA基因序列突变位点设计引物和探针,分别检测淋球菌对喹诺酮类药物和大观霉素的耐药性;根据淋球菌和解脲脲原体的共同tetM基因序列设计引物和探针,检测对四环素的耐药性。制备芯片,对152份泌尿生殖道拭子提取DNA进行7重PCR扩增,并且Cy5荧光标记包含上述基因序列的目的DNA片段,与固定在芯片上的探针杂交,芯片信号分析系统Scanarray 4000在635nm处扫描并分析结果,并与临床检查结果比较。结果 DNA芯片敏感性是0.01fg质粒DNA。152份泌尿生殖道炎症拭子其病原体种类及其耐药类型全部可用DNA芯片检测出来,与目前临床检查结果有较好的一致性(K>0.8)。结论 研制的DNA芯片结合7重PCR方法快速检测泌尿生殖道淋球菌、沙眼衣原体和解脲脲原体3种病原体及其对喹诺酮类药物、大观霉素和四环素的耐药类型,具有快速、高特异性和高灵敏度,可以应用于临床检测。

关键词: 奈瑟球菌, 淋病, 衣原体, 沙眼, 尿素支原体, 尿素分解, 寡核苷酸序列分析, 微生物敏感性试验

Abstract: Objective To develop a DNA chip combined with multiplex PCR for detecting 3 prevalent pathogens of urogenital infections and their drug-resistance. Methods The primers and probes targeting the conservative sequences of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum were designed for the detection of the pathogens. And the primers and probes targeting gyrA, parC, 16 srRNA and tetM sequences were designed for detecting fluoroquinolone, spectinomycin, tetracycline-resistant Neisseria gonorrhoeae and tetracycline-resistant Ureaplasma urealyticum. These probes were used for fabricating a DNA chip. The total DNA was isolated from 152 genital clinical isolates, and the target DNA was amplified by multiplex PCR, labeled with Cy5 fluorescence, and then hybridized with the DNA chip. The scanarray 4 000 system was used for scanning and analysing the results. Results The sensitivity of the DNA chip is 0.01 fg plasmid DNA. The results detected by the DNA chip were completely consistent with those of clinical routine tests (k>0.8). Conclusions This DNA chip combined with multiplex PCR is a rapid diagnostic assay with high specificity and sensitivity for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma Urealyticum and their drug-resistance, and may be applied in the diagnosis of urogenital infections.

Key words: Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Oligonucleotide array sequence analysis, Microbial sensitivity tests