本文選題：瘧疾 + 環(huán)介導等溫擴增　； 參考：《東華大學》2012年碩士論文

【摘要】：蚊媒傳播的瘧疾是一種嚴重危害人類健康和生命的熱帶傳染病,在全世界人群中具有很高的發(fā)病率和致死率,已成為全球最重要的公共衛(wèi)生問題之一,世界衛(wèi)生組織已將瘧疾和艾滋病、結(jié)核病一起列為全球三大公共衛(wèi)生問題。準確診斷一直是瘧疾防治工作的一個重要環(huán)節(jié),顯微鏡檢查作為金標準目前仍是診斷瘧疾的主要手段。但是鏡檢需要專業(yè)的實驗操作技術和豐富的經(jīng)驗,如今有經(jīng)驗的鏡檢人員越來越缺乏；PCR檢測是另一種廣泛使用的方法,盡管其敏感性高,但耗費時間長且需要特殊儀器、檢測成本較高。因此迫切需要一種敏感、特異和簡便的瘧原蟲檢測方法。 目的運用環(huán)介導等溫擴增技術(Loop-mediated Isothermal Amplification,LAMP)建立一種快速、簡便、敏感度高的瘧原蟲檢測方法。 方法本研究確定瘧原蟲環(huán)子孢子蛋白(CSP)基因為靶基因,設計合成特異性LAMP引物。構(gòu)建間日瘧原蟲18S核糖體小亞基ssRNA特異性基因片段的重組質(zhì)粒DNA (Pv-rDNA)作為陽性對照。優(yōu)化Mg2+濃度、dNTPs濃度、Bst DNA聚合酶添加量、反應溫度、時間以及設計引物缺省試驗。評估優(yōu)化后的LAMP反應的特異性和靈敏性。檢測133份患者血樣,以顯微鏡檢方法為金標準,比較LAMP和多重PCR法檢測間日瘧原蟲的敏感性和特異性。初步應用LAMP法檢測斯氏按蚊體內(nèi)鼠約氏瘧原蟲,對其特異性和靈敏性進行評估。 結(jié)果建立了采用LAMP技術快速檢測人體內(nèi)間日瘧原蟲的方法,LAMP法檢測重組質(zhì)粒DNA (Pv-rDNA)的靈敏度達到10-10,為傳統(tǒng)PCR方法的100倍。鏡檢確診的68例間日瘧患者,43例惡性瘧患者和22例非瘧疾患者中,LAMP法和多重PCR檢測間日瘧原蟲的敏感性為98.53%和97.06%,兩法檢測靈敏度一致；特異性為86.15%和100%,LAMP法低于多重PCR法。LAMP法的陽性預測值和陰性預測值分別為88.16%和98.25%,多重PCR的陽性預測值和陰性預測值分別為100%和97.01%。選擇斯氏瘧原蟲唾液腺子孢子微線體SPECT2作為靶基因合成LAMP反應引物,用該法初步應用于斯氏按蚊體內(nèi)鼠約氏瘧原蟲的檢測,特異性好,敏感性為能檢測出在80只陰性斯氏按蚊中有1只約氏瘧原蟲陽性的斯氏按蚊的混合樣本。 結(jié)論本研究建立的人體內(nèi)間日瘧原蟲LAMP檢測方法具有快速簡便、敏感性高、設備要求低、成本低廉的特點,具有較好的現(xiàn)場應用前景；LAMP法初步應用于斯氏按蚊體內(nèi)鼠約氏瘧原蟲的檢測,特異性和敏感性均較好,為進一步研究蚊體內(nèi)瘧原蟲子孢子LAMP法檢測奠定基礎。
[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.
【學位授予單位】：東華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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