几种性传播疾病病原体检测芯片的制备  

Preparation of oligonucleotide microarray for detecting pathogens responsible fo r sexually transmitted diseases

作　　者：石岗[1] 文思远[1] 陈苏红[1] 王升启[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2004年第5期405-409,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金重点项目 (3 98890 0 1)

摘　　要：目的 :为了同时多样本检测和鉴别淋病奈瑟球菌、沙眼衣原体和解脲脲支原体 3种重要的性传播疾病病原体 ,制备了寡核苷酸检测芯片。方法 :针对 3种病原体和荧光素酶基因设计特异的引物和寡核苷酸探针 ,采用硫代和氨基双功能探针修饰技术制备寡核苷酸芯片 ,以荧光标记多重不对称PCR技术为基础 ,通过将单链PCR产物与芯片杂交实现对性传播疾病病原体的检测。结果 :对 10种与待检病原体无关的菌及定量有限稀释的荧光素酶和 3种病原体基因质粒模板进行芯片检测 ,结果表明芯片对待检病原体特异 ,其检测 4种基因的灵敏度均为 5×10 3 拷贝质粒。对 2 4份性传播疾病患者标本进行芯片检测 ,沙眼衣原体感染率为 10 0 % ,与淋病奈瑟球菌混合感染率为 83.3% (2 0 / 2 4 ) ,与传统PCR诊断结果完全一致。在 2 4份标本中 ,淋病奈瑟球菌、沙眼衣原体和解脲脲支原体三重感染病例芯片诊断为 3例 ,混合感染率为 12 .5 % (3/ 2 4 ) ;而传统PCR诊断为 4例 ,混合感染率为 16 .7% (4/2 4 ) ,两种方法的符合率为 75 %。结论 :该芯片是一种可靠检测 3种病原体的方法 ,它可快速提供有关患者混合感染的情况 ,因而为指导个性化治疗提供及时可靠的诊断依据。Objective: To prepare a specific oligonucleotide microarray for detec ting and identifying Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) in one reaction.Methods: The specific primers for NG, CT, UU and lucifer as e (Luc) used as an internal control were designed by the software Primer 5.0 and the specific oligonucleotide probes for NG, CT, UU and Luc as well as lectin (L ec) used as a negative control were designed by the software DNASIS 5.0. The rev erse primers specific for NG, CT, UU and Luc were labeled with fluorescein (Cy 3) at 5′-terminal. The specific probes for NG, CT, UU, Luc and the negative c ont rol were synthesized with a poly (A) spacer consisting of five adenine thiophosp hates and a free amino group linked at the 3′-terminal of each probe. The olig onucleotide microarray was prepared by mixing each oligonucleotide probe at a co ncentration of 100 μmol/L with a spotting solution at a ratio of 1∶1 and spotting the mixture onto an aldehyde-derived glass slide. A multiplex asym metric PCR was used to produce NG, CT, UU and Luc-specific single-stranded s eg ments in a PCR reaction at a ratio of 10∶1 for the concentration of ea ch Cy3-labeled reverse primer to that of each forward primer. The Cy3-labeled PCR products were mixed with a hybridization solution and transferred to a hybri dization area of the microarray. After incubation at 42℃ for 1h, the slide was scanned using GenePix 4000 and the STD pathogens were determined by the signals and their intensities of each probe on the slide. Results: Ten species of strains irrelative to NG, CT, UU w ere investigated using the microarray and no specific signals were detected by t he microarray except the internal control signals, suggesting that the microarr a y be specific for three STD pathogens. Pooled templates consisting of NG, CT, U U and Luc plasmids at a series of concentrations from 5×10 5copies to 5×10 2copies per reaction were subjected to the microarray analysis an d the m

关 键 词：性传播疾病 寡核苷酸芯片 寡核苷酸探针 淋病奈瑟球菌 沙眼衣原体 解脲脲支原体 

分 类 号：R759[医药卫生—皮肤病学与性病学]