问号钩端螺旋体检测基因芯片的研制

目的　制备一种用于快速检测、鉴定问号钩端螺旋体的DNA微阵列芯片。方法　从Genbank中选取钩体 2 3SrDNA序列 ,设计一对种特异性引物和DNA探针 ,通过Blast的基因同源性比较 ,验证引物和探针的通用性与特异性。将合成的探针点样到玻片上制备成基因芯片。用Cy3标记引物 ,通过不对称PCR反应获取荧光素标记的靶序列 ,然后与制备的芯片杂交。结果　设计的引物与探针仅同时与问号钩体 2 3srDNA完全同源。该引物可扩增我国 18个血清群的 2 5株钩体 ,均出现单一 4 82bp的扩增产物 ,而双曲钩体及其他螺旋体、病原、空白对照均无任何DNA扩增条带。芯片杂交结果与常规PCR方法结果一致 ,敏感性高于PCR。结论　基因芯片可以快速、灵敏、特异地检测问号钩端螺旋体。

Development of a DNA chip for detection of Leptospira interrogans

To develop a specific and sensitive DNA chip for detection of L.interrogans,primers and specific probes were designed from 23s rDNA sequence of L.interrogans,and their generality and specificity were verified by comparison of gene homology.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 DNA chip for detection of L.interrogans strains and others.It was found that the designed primers and probes were completely homologous with all the sequences of 23s rDNAs of L. interrogans from Genbank.A specific amplification product with 482 bp in length could be found from 25 serovar L.interrogans by conventional PCR,and conversely for the other microorganisms.Positive signal were all displayed on the microchips in L.interrogans strains,but other pathogens failed to yield any signal under the same conditions.The sensitivity of detection by the chip was shown to be higher than PCR amplification.This DNA chip is more specific and sensitive than other detection method for L.interrogans.