基因芯片技术在钩端螺旋体病传染源监测中的应用

为了将合成的核酸探针点样到玻片 上制备成基因芯片,先用常规PCR扩增动物 组织中钩端螺旋体特异片段,再用Cy3标记 引物,通过不对称PCR获取荧光素标记的靶 序列,然后与制备的芯片杂交,扫描仪记录结 果。同时,以PCR技术为对照,进一步观察 基因芯片技术检测动物传染源中钩端螺旋体 的特异性、敏感性及可行性。常规PCR扩增 问号钩端螺旋体阳性标本,出现单一482bp 的扩增产物,而双曲钩端螺旋体及其他螺旋 体、病原、正常动物组织均无任何DNA扩增 条带,芯片杂交结果与常规PCR方法结果一 致。用芯片和PCR对动物组织中不同浓度 钩端螺旋体的检测,最低检测浓度分别为10 条和100条钩端螺旋体,芯片检测的敏感性 高于常规PCR法。用基因芯片与PCR分别 对标本进行检测,5只人工感染的豚鼠肾、 肝、心、血等组织的检测结果均为阳性;28份 疫区野鼠肾标本阳性结果分别为8份和4 份,10份可疑病猪肾标本阳性结果分别7份 和5份;5份非疫区野鼠肾、5份正常猪肾标 本以及其他微生物的检测结果均为阴性。结 果表明,基因芯片技术可快速、灵敏、特异地 检测动物传染源中问号钩端螺旋体。

Detecting Animal Infection Source of Leptospirosis By Genechip Technique

Different oligonucleotide probes or controls were spotted on glass slides in quarteticate with a low density array format.The fluorescent-labeled PCR products were prepared by incorporation of Cy3-labeled primer during PCR amplification and were hybridized to the genechips for detection of Leptospira interrogans strains and others.Their sensibility,specificity and feasibility were compared with conventional PCR.A specific amplification product with 482 bp length could be found from L.interrogans by conventional PCR,and conversely for the other microorganisms and normal animal tissue.The results showed no difference between genechip hybridization and conventional PCR.Detecting different concentration of L.interrogans blended into normal animal tissue with genechip and PCR,their minimum detecting concentration were 10 and 100 leptospiras, respectively. The sensibility of genechip was higher than that of routine PCR.Detecting the kidney,liver,heart and blood of 5 infected guinea pigs with genechip and PCR,their results were positive;Detecting 28 rat kidney specimens from epidemic area by the two methods,8 and 4 were positive, respectively. Detecting 10 kidney specimens of suspicious infected pigs,7 and 5 were positive, respectively.Detecting 5 rat kidney specimens from non-epidemic area, 5 normal kidney specimens and other microbe,the results were negative.These results revealed that genechip technique could rapidly, sensitively,specifically detect L.interrogans in animal source of infection.