7种立克次体甄别检测基因芯片方法的建立

DNA microarray for simultaneous screening and detection of seven rickettsia

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 develop a chemiluminescence（ CL） imaging DNA microarray method for simultaneous detection of seven rickettsiae. Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes. The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae. The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method,and then hybridized with microarray that was scanned after washing and chemiluminescence coloration,before the results were analyzed. Facilitated by the optimization of the multiplex PCR system,hybridization,and chemiluminescence imagination,we evaluated the specificity,sensitivity and reproducibility of the chip. The serial diluted nucleic acid of Rickettsia mooseri was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods. Double-blind simulated samples were prepared to further evaluate the accuracy of the detection methods. Results One universal primer,four specific primers,one universal probe,and nine specific probes were selected. This DNA microarray demonstrated high specificity and sensitivity. The detection sensitivity of plasmid DNA and double-blind simulated sample DNA was 1. 5 × 10^2-3 × 10^3 copies per reaction and 103- 104 copies / μl. The detection results of real-time PCR method was consistent with the microarray,and the microarray possessed 10 fold lower detection sensitivities than the real-time PCR method. The coincidence rate of double-blind simulated sample detection was 100%. Conclusion A chemiluminescence imaging DNA microarray method for simultaneous detection of seven rickettsiae is established successfully,which can serve as a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsiae.