论文部分内容阅读
目的建立一种能同时、快速、准确地检测包括:麻疹病毒、风疹病毒、肠道病毒71型、水痘带状疱疹病毒、登革热病毒、人类小DNA病毒B19、柯萨奇病毒A16型、A组β型酿脓链球菌(溶血性链球菌)、伤寒沙门菌,9种发热伴出疹病原体的化学发光基因芯片的方法。方法根据9种病原体特异基因中的高度保守区序列设计引物与探针,进行多重不对称PCR扩增,将带有标记的扩增产物与带有特异性探针的芯片杂交,经洗涤、化学发光显色后进行结果分析。经多重PCR体系、杂交反应和化学发光检测条件的优化,评价该芯片的特异性、灵敏度和重复性。实时荧光PCR法与芯片法分别检测肠道病毒EV71梯度稀释的核酸,比较两种方法的灵敏度。结果共筛选出9对特异性扩增引物和11条特异性检测探针。该芯片具有良好的特异性、灵敏度和重复性。质粒参考品和体外转录RNA参考品的最低检测限为3×10~3拷贝/反应,芯片法的灵敏度为实时荧光PCR法的1/10。检测74例临床样本的灵敏度为95%,特异性为85.7%,总符合率为93.2%。结论建立了一种可同时检测9种发热伴出疹病原体的化学发光基因芯片的检测方法,为发热伴出疹的临床诊断和流行病学调查提供了一种高通量的筛查手段。
OBJECTIVE To establish a rapid, accurate and rapid detection method for measles virus, rubella virus, enterovirus 71, varicella-zoster virus, dengue virus, human small DNA virus B19, coxsackievirus A16, beta-type Streptococcus pyogenes (hemolytic streptococcus), Salmonella typhi, nine kinds of fever with rash pathogen chemiluminescence gene chip method. Methods Primers and probes were designed based on the sequences of highly conserved regions of 9 pathogen-specific genes. Multiple asymmetric PCR amplification was carried out. The labeled products were hybridized with the probes with specific probes. After washing, Luminescence color after the results of analysis. The specificity, sensitivity and reproducibility of the chip were evaluated by multiplex PCR, hybridization and chemiluminescence detection. Real-time fluorescence PCR and microarray were used to detect EV71 gradient-diluted nucleic acid respectively, and the sensitivity of the two methods was compared. Results Nine pairs of specific amplification primers and 11 specific detection probes were screened out. The chip has good specificity, sensitivity and repeatability. The minimum detection limit of plasmid reference and in vitro transcription RNA reference is 3 × 10 ~ 3 copies / reaction, and the sensitivity of chip method is 1/10 of that of real-time PCR. Sensitivity of the detection of 74 clinical samples was 95%, specificity was 85.7%, the total coincidence rate was 93.2%. Conclusion A chemiluminescent microarray detection method capable of detecting 9 pathogens causing fever and rash at the same time was established, which provided a high-throughput screening method for the clinical diagnosis and epidemiological investigation of fever and rash.