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目的 :以 1 6SrDNA为对象 ,生物芯片微阵列技术为平台 ,建立一种能在未知标本中快速检测 6种立克次体的方法。方法 :针对每种立克次体 1 6SrDNA的可变区 ,分别设计和合成 4条寡核苷酸探针。利用微阵列技术构建立克次体检测用生物芯片。待测立克次体染色体DNA用 1 6SrRNA通用引物扩增掺入荧光素 ,然后与芯片杂交。利用各种立克次体的 4条特异性探针是否全部出现杂交信号来做出判断。结果 :以寡核苷酸为探针的生物芯片系统可以实现 6种立克次体的检测。对伯氏考克斯体、汉氏巴尔通体、恙虫病东方体 ,本系统可以将其鉴定到种或属的位置。从第 4号探针的不同可以将普氏立克次体和立氏立克次体这两个群区分开来。犬埃立克体检测始终为阴性 ,可能和没有合适的标本有关。从接到样品到判读出结果 ,整个检测过程大约需要 4 .5h。敏感性检测结果表明本法比PCR_电泳法敏感 1 0倍。结论 :利用 1 6SrDNA生物芯片微阵列方法可以快速检测 6种立克次体
OBJECTIVE: To establish a method for rapidly detecting six species of Rickettsia in unknown samples using 16SrDNA as a target and a biochip microarray technology as a platform. Methods: For each Rickettsia 16SrDNA variable region, four oligonucleotide probes were designed and synthesized. Construction of Rickettsia biochip using microarray technology. The Rickettsia chromosomal DNA to be tested is amplified with fluorescein by a 16S rRNA universal primer followed by hybridization to the chip. Judgment was made on whether all four specific probes of various rickettsia hybridization signals appear. Results: Oligonucleotide-based biochip systems were able to detect 6 Rickettsia species. On the Cox’s body, Bartlett’s body, tsutsugamushi oriental body, the system can be identified to species or genera position. From the difference of the No. 4 probe, the two groups of Rickettsia and Rickettsia can be distinguished. Canine ehrlichiosis test is always negative, may be related to the absence of suitable specimens. From receiving the sample to interpreting the result, the whole test takes about 4.5 h. Sensitivity test results show that this method is 10 times sensitive than PCR_ electrophoresis. Conclusion: The 16S rDNA biochip microarray method can be used to detect 6 Rickettsia