环介导等温扩增技术检测疟原虫方法的建立及其应用

发布时间：2018-06-08 16:41

本文选题：疟疾 + 环介导等温扩增　；参考：《东华大学》2012年硕士论文

【摘要】：蚊媒传播的疟疾是一种严重危害人类健康和生命的热带传染病,在全世界人群中具有很高的发病率和致死率,已成为全球最重要的公共卫生问题之一,世界卫生组织已将疟疾和艾滋病、结核病一起列为全球三大公共卫生问题。准确诊断一直是疟疾防治工作的一个重要环节,显微镜检查作为金标准目前仍是诊断疟疾的主要手段。但是镜检需要专业的实验操作技术和丰富的经验,如今有经验的镜检人员越来越缺乏；PCR检测是另一种广泛使用的方法,尽管其敏感性高,但耗费时间长且需要特殊仪器、检测成本较高。因此迫切需要一种敏感、特异和简便的疟原虫检测方法。目的运用环介导等温扩增技术(Loop-mediated Isothermal Amplification,LAMP)建立一种快速、简便、敏感度高的疟原虫检测方法。方法本研究确定疟原虫环子孢子蛋白(CSP)基因为靶基因,设计合成特异性LAMP引物。构建间日疟原虫18S核糖体小亚基ssRNA特异性基因片段的重组质粒DNA (Pv-rDNA)作为阳性对照。优化Mg2+浓度、dNTPs浓度、Bst DNA聚合酶添加量、反应温度、时间以及设计引物缺省试验。评估优化后的LAMP反应的特异性和灵敏性。检测133份患者血样,以显微镜检方法为金标准,比较LAMP和多重PCR法检测间日疟原虫的敏感性和特异性。初步应用LAMP法检测斯氏按蚊体内鼠约氏疟原虫,对其特异性和灵敏性进行评估。结果建立了采用LAMP技术快速检测人体内间日疟原虫的方法,LAMP法检测重组质粒DNA (Pv-rDNA)的灵敏度达到10-10,为传统PCR方法的100倍。镜检确诊的68例间日疟患者,43例恶性疟患者和22例非疟疾患者中,LAMP法和多重PCR检测间日疟原虫的敏感性为98.53%和97.06%,两法检测灵敏度一致；特异性为86.15%和100%,LAMP法低于多重PCR法。LAMP法的阳性预测值和阴性预测值分别为88.16%和98.25%,多重PCR的阳性预测值和阴性预测值分别为100%和97.01%。选择斯氏疟原虫唾液腺子孢子微线体SPECT2作为靶基因合成LAMP反应引物,用该法初步应用于斯氏按蚊体内鼠约氏疟原虫的检测,特异性好,敏感性为能检测出在80只阴性斯氏按蚊中有1只约氏疟原虫阳性的斯氏按蚊的混合样本。结论本研究建立的人体内间日疟原虫LAMP检测方法具有快速简便、敏感性高、设备要求低、成本低廉的特点,具有较好的现场应用前景；LAMP法初步应用于斯氏按蚊体内鼠约氏疟原虫的检测,特异性和敏感性均较好,为进一步研究蚊体内疟原虫子孢子LAMP法检测奠定基础。
[Abstract]:Mosquito-borne malaria is a tropical infectious disease that seriously endangers human health and life. It has a high incidence of morbidity and mortality among people throughout the world and has become one of the most important public health problems in the world. The World Health Organization has listed malaria, AIDS and tuberculosis as three major public health problems in the world. Accurate diagnosis has always been an important link in malaria control. Microscope, as a gold standard, is still the main method of malaria diagnosis. But microscopic examination requires professional experimental techniques and rich experience. Nowadays, experienced mirror examiners are increasingly lacking in PCR detection, which is another widely used method. Although its sensitivity is high, it takes a long time and requires special instruments. The cost of detection is high. Therefore, there is an urgent need for a sensitive, specific and simple method for the detection of Plasmodium falciparum. Objective to establish a rapid and simple method for the detection of Plasmodium falciparum by Loop-mediated Isothermal Amplification (Lamp). Methods in this study, specific lamp primers were designed and synthesized to identify the CSP gene of Plasmodium circumsporum protein (CSP) as the target gene of Plasmodium falciparum. The recombinant plasmid Pv-rDNA of 18s ribosomal small subunit ssRNA specific gene fragment of Plasmodium vivax was constructed as positive control. Optimization of Mg2 concentration and dNTPs concentration of BST DNA polymerase, reaction temperature, reaction time and default primer design test. To evaluate the specificity and sensitivity of the optimized lamp reaction. The sensitivity and specificity of lamp and multiplex PCR for the detection of Plasmodium vivax were compared. The method of lamp was used to detect Plasmodium yoelii in Anopheles stephensi. Results the sensitivity of lamp method for rapid detection of Plasmodium vivax was 10-10, 100 times higher than that of traditional PCR. The sensitivity of lamp and multiplex PCR for the detection of Plasmodium vivax was 98.53% and 97.06% respectively in 43 patients with falciparum malaria and 22 patients without malaria. The sensitivity of the two methods was the same. The specificity was 86.15% and 100% respectively. The positive predictive value and negative predictive value of multiplex PCR were 88.16% and 98.25, respectively. The positive and negative predictive values of multiplex PCR were 100% and 97.01%, respectively. SPECT2 was selected as the target gene to synthesize lamp reaction primer. The method was applied to the detection of Plasmodium yoelii in Anopheles stephensi with good specificity. The sensitivity was to detect a mixed sample of Anopheles stephensi that was positive for Plasmodium yoelii in 80 negative Anopheles stephensi. Conclusion the lamp method developed in this study is rapid, simple and sensitive to the detection of Plasmodium vivax. The equipment has the characteristics of low requirement and low cost. It has a good prospect of field application. The lamp method is applied to the detection of Plasmodium yoelii in Anopheles stephensi. The specificity and sensitivity of lamp method are good. For further study on the detection of Plasmodium falciparum sporozoite lamp method in mosquito.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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