两种传染病病原体复合探针荧光PCR检测方法的建立

发布时间：2019-05-27 09:41

【摘要】：目的:感染性疾病是人类进入21世纪以来,对人类健康安全危害极大的疾患。它具有传播速度快、感染性强、危害大的特点。经呼吸道传播和蚊虫叮咬传播是两种主要的传播途径。如曾引起世界范围内关注的传染性非典型肺炎(SARS)、人禽流感、甲型H1N1流感以及黄热病等。因此,对传染性致病病毒进行快速准确的诊断是保障公众健康和防控疫情的关键之一。然而,现在大部分临床卫生医疗机构使用的传统微生物检验法涉及培养、分离等步骤,操作繁琐、检测周期长。本研究的目的是建立一种检测经呼吸道传播和蚊虫叮咬传播两种病毒的快速、特异的诊断方法(PCR荧光探针法)。避免一般PCR易出现假阳性、交叉污染等问题,从而能对两种病毒进行快速又准确的检测,进而更好地指导临床治疗。方法:首先,阅读有关人类副流感病毒和寨卡病毒的相关文献,确定本研究要检测的特异基因,以此为依据设计引物和复合探针,然后以临床样本为模板制备质粒参考品。其次,通过优化PCR反应体系和反应条件来确定引物探针的最佳浓度和比例。最后,评价该检测方法的灵敏性、重复性和特异性;选择样本核酸提取效率最高的试剂;模板的最佳用量;不同PCR仪器对检测结果的影响以及临床样本的检测。结果:查阅文献后确定人类副流感病毒的特异基因为HN基因。确定了PCR反应的最佳反应体系和反应条件,其中HPIV1和HPIV3的上、下游引物在体系中的终浓度为0.4μmol/L,探针在体系中的终浓度为0.2μmol/L;HPIV2和内标的上、下游引物在体系中的终浓度为0.15μmol/L,探针的终浓度为0.06μmol/L;其HPIV1、HPIV2和HPIV3探针的荧光探针与淬灭探针的比例为F∶Q=1∶2。退火温度为58℃,退火时间为30s。最终该试剂盒的各项性能指标显示该试剂盒最低可检出不低于5×102 copies/μl浓度的样本,重复检测10次结果表明其CV值小于5%,对10个种属相近的病原体检测并没有扩增,说明该试剂盒特异性良好。且两种样本核酸提取试剂效果相当,样本用量选取为3μl。三种不同机型检测结果表明不同机型对实验结果影响不大。临床样本检测结果符合率为100%。确定寨卡病毒的特异基因为NS5基因。引物探针性能评价结果表明其符合引物探针设计原则。检测灵敏度可达到5×102 copies/μl,重复检测10次结果表明其CV值小于10%。建立的检测方法与其他种属无交叉。说明本试剂盒具有良好的灵敏性、精密性和特异性。结论:本研究成功建立了一种可同时检测人类副流感病毒3个亚型和寨卡病毒的诊断方法(PCR荧光探针法)。该方法灵敏度高、重复性好、特异性良好。为人类副流感病毒和寨卡病毒提供了一种特异、安全、快速、准确的检测方法,可以用于疾病的早期诊断,以指导及时、准确地采取防护和治疗措施。
[Abstract]:Objective: infectious disease is a disease that is very harmful to human health and safety since the beginning of the 21 st century. It has the characteristics of fast transmission, strong infection and great harm. Respiratory tract transmission and mosquito bite transmission are the two main routes of transmission. Such as infectious atypical pneumonia (SARS), avian influenza, H1N1 influenza and yellow fever, which have attracted worldwide attention. Therefore, the rapid and accurate diagnosis of infectious pathogenic virus is one of the keys to ensure public health and prevent and control the epidemic situation. However, most of the traditional microbiological testing methods used in clinical health and medical institutions involve culture, separation and other steps, the operation is tedious and the detection cycle is long. The purpose of this study was to establish a rapid and specific diagnostic method for the detection of respiratory tract transmission and mosquito bite transmission (PCR fluorescence probe method). Avoid false positive, cross-contamination and other problems in general PCR, so that the two viruses can be detected quickly and accurately, and then better guide clinical treatment. Methods: firstly, the literature on human parainfluenza virus and Zika virus was read, the specific genes to be detected in this study were determined, and primers and composite probes were designed on the basis of which primers and composite probes were designed, and then the plasmid reference materials were prepared by using clinical samples as templates. Secondly, the optimum concentration and proportion of primer probe were determined by optimizing PCR reaction system and reaction conditions. Finally, the sensitivity, reproducibility and specificity of the detection method were evaluated, the reagent with the highest nucleic acid extraction efficiency was selected, the optimum dosage of template, the influence of different PCR instruments on the detection results and the detection of clinical samples were selected. Results: after consulting the literature, the specific gene of human parainfluenza virus was identified as HN gene. The optimum reaction system and reaction conditions of PCR reaction were determined, in which the final concentration of downstream primers in the system of HPIV1 and HPIV3 was 0.4 渭 mol / L, and the final concentration of probe in the system was 0.2 渭 mol / L. The final concentration of upstream and downstream primers in HPIV2 and internal standard was 0.15 渭 mol / L, and the final concentration of probe was 0.06 渭 mol / L, and the ratio of fluorescence probe to quenching probe of HPIV1,HPIV2 and HPIV3 probe was F 鈮，

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