中华皮肤科杂志

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皮肤分枝杆菌感染微孔板反向杂交检测法的研究

王洪生, 李晓杰, 吴勤学, 崔盘根, 刘训荃   

  1. 中国医学科学院、中国协和医科大学皮肤病研究所, 南京, 210042
  • 收稿日期:2006-06-30 出版日期:2007-06-15 发布日期:2007-06-15
  • 通讯作者: 吴勤学,email:whs33@vip.sina.com E-mail:whs33@vip.sina.com
  • 基金资助:
    江苏省自然科学基金(BK2006015)

Development of a PCR-microplate reverse hybridization method for the detection and identification of several species of mycobacteria

WANG Hong-sheng, LI Xiao-jie, WU Qin-xue, CUI Pan-gen, LIU Xun-quan   

  1. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical Col-lege, Nanjing 210042, China
  • Received:2006-06-30 Online:2007-06-15 Published:2007-06-15

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摘要： 目的 探讨微孔板反向分子杂交技术与PCR-ELISA相结合的“拟芯片”法检测和鉴定几种常见的皮肤分枝杆菌感染的方法。方法 首先对5种分枝杆菌标准菌株(结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌和偶遇分枝杆菌)16s rRNA DNA进行PCR扩增获得其PCR产物:其次对"拟芯片"法反应条件进行优化;并对其敏感性、特异性进行检测;再用建立的方法对9例临床分离株进行检测和鉴定。结果 "拟芯片"法反应条件优化结果为:探针包被浓度为100 nmol/mL,探针包被时间为15 min,杂交的温度为55℃,杂交时间为45 min,辣根过氧化物酶标记的亲和素的稀释度为1:800;"拟芯片"法敏感性为1×101～1×102个菌细胞/mL;特异性较好,各菌探针与其他分枝杆菌间无交叉。结论 "拟芯片"法是快速、敏感及特异的皮肤分枝杆菌感染诊断方法之一。

关键词: 寡核苷酸序列分析, 分枝杆菌属, 双杂交系统技术

Abstract: Objective To develop a PCR-microplate reverse hybridization("simulant chip") method for the detection and identification of several species of mycobacteria in cutaneous infection.Methods DNA was extracted from reference strains of 5 species(M.tuberculosis,M.avium,M.intracellulare,M. kansasii,M.fortuitum)of mycobacteria,and amplified by PCR targeting 16s rRNA.The coating concentra-tion and time of probes in wells,time and temperature of hybridization,and dilution fold of avidin-horseradish peroxidase were optimized.Five standard and 9 clinical strains of mycobacterium were dectected by"simulant chip"according to the optimized condition to test the sensitivity and specificity of this method.Results The followings were the optimum values:of concentration of probe,100 nmol/ml,for coated time,15 minutes, time and temperature of hybridization,45 minutes and 55℃,respectively,and of dilution fold of avidin-horseradish peroxidase,1:800.The detection limit of"simulant chip"was 1×101～1×102 cells/mL for these mycobacteria.No cross reaction was observed for the probes and other species of mycobacterium.Conclusion The"simulant chip"is a rapid,sensitive,specific and convenient method for the diagnosis of cutaneous mycobacterium infection.

Key words: Mycobacterium, Two-hybrid system techniques, Oligonucleotide array sequence analysis