7种立克次体甄别检测基因芯片方法的建立

发布时间：2018-01-28 06:46

本文关键词： 立克次体属基因芯片多重PCR 化学发光测定法　出处：《军事医学》2015年07期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to establish a chemiluminescence gene chip method for simultaneous detection of seven Rickettsia species. Methods Primer and probe were designed according to the sequences of 7 Rickettsia strains published by NCBI. The target gene fragment of Rickettsia was amplified by multiple asymmetric PCR. The labeled product was hybridized with the probe on the gene chip and cleaned. The specificity and sensitivity of the chip were evaluated under the conditions of optimized multiplex PCR system, hybridization reaction and chemiluminescence detection. Repeatability. Real-time fluorescent PCR and chip method were used to detect the gradient dilution nucleic acid of Rickettsia Morse respectively. The sensitivity of the two methods was compared. A double-blind simulated sample was prepared. Results A total of 1 pair of universal primers, 4 pairs of specific primers and 1 common probe of Rickettsia were screened. The sensitivity of the microarray for plasmid DNA detection was 1.5 脳 10 ~ 2 ~ 3 脳 10 ~ 3 copies / reaction. The sensitivity of detecting simulated samples is 103 ~ 104copies / 渭 l. The results of real-time fluorescence PCR method are consistent with those of chip method. The sensitivity of real-time fluorescence PCR method is 10 times higher than that of chip method. The coincidence rate of double-blind analog sample detection is 1000.Conclusion A chemiluminescence gene chip method for simultaneous detection of seven Rickettsiae has been successfully established. It provides a new high-throughput detection method for the clinical diagnosis and epidemiological investigation of Rickettsia disease.
【作者单位】：河南中医学院;军事医学科学院放射与辐射医学研究所;
【基金】：广东省科技重大专项资助项目(2012A080203005)
【分类号】：R440;R513
【正文快照】： 立克次体(rickettsiae)除个别外是一类专性细胞内寄生的革兰阴性菌[1],而引起的立克次体病是一类重要的人畜共患自然疫源性疾病,在世界范围内呈散发性和季节性流行。许多节肢动物,如虱、蚤、蜱、螨等均可作为立克次体感染的传播媒介[2]。随着国际旅游业的发展,立克次体病被认

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