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目的建立一种能同时检测7种立克次体的化学发光基因芯片法。方法根据NCBI公开发表的7种立克次体的序列设计引物和探针,制备立克次体甄别检测基因芯片。利用多重不对称PCR法扩增立克次体靶基因片段,标记的产物与基因芯片上的探针杂交,经清洗、化学发光显色后进行结果分析。在优化的多重PCR体系、杂交反应和化学发光检测条件下,评价芯片的特异性、灵敏度、重复性。用实时荧光PCR法与芯片法分别检测莫氏立克次体梯度稀释的核酸,比较两种方法的灵敏度。制备双盲模拟样本,进一步评价芯片方法的准确性。结果该研究共筛选出1对通用引物、4对特异性引物和1条立克次体属通用探针、9条特异性检测探针。该芯片检测质粒DNA的灵敏度为1.5×102~3×103拷贝/反应,检测模拟样本的灵敏度为103~104拷贝/μl。实时荧光PCR法与芯片法检测结果一致,实时荧光PCR法比芯片法灵敏度高10倍。双盲模拟样本检测符合率为100%。结论成功建立了可同时检测7种立克次体的化学发光基因芯片检测方法,为立克次体病的临床诊断和流行病学调查提供了一种新的高通量检测手段。
Objective To establish a chemiluminescence microarray method capable of simultaneously detecting seven species of rickettsia. Methods Primers and probes were designed according to the sequences of seven Rickettsia published by NCBI to prepare Rickettsia screening and detection gene chips. The Rickettsia target gene fragment was amplified by multiple asymmetric PCR. The labeled product was hybridized with the probe on the gene chip, and the result was analyzed by washing and chemiluminescence. In the optimized multiplex PCR system, hybridization reaction and chemiluminescence detection conditions, evaluate the chip specificity, sensitivity, repeatability. Real-time fluorescence PCR and chip were used to detect the gradient dilution of Rickettsia monodon, and the sensitivity of the two methods were compared. Double-blind simulation samples were prepared to further evaluate the accuracy of the chip method. Results A total of 1 universal primer, 4 pairs of specific primers and 1 rickettsia common probe were selected and 9 specific detection probes were screened out. The sensitivity of this chip for detecting plasmid DNA is 1.5 × 102 ~ 3 × 103 copies / reaction, and the sensitivity of the test sample is 103 ~ 104 copies / μl. Real-time fluorescent PCR method and the chip test results are consistent, real-time fluorescent PCR method than the chip sensitivity 10 times. Double-blind simulation sample detection coincidence rate of 100%. Conclusion The chemiluminescence microarray detection method, which can detect seven kinds of rickettsia at the same time, has been established successfully. It provides a new high-throughput detection method for the clinical diagnosis and epidemiological investigation of rickettsial disease.