本文選題：瘧疾 + 環(huán)介導(dǎo)等溫?cái)U(kuò)增　； 參考：《東華大學(xué)》2012年碩士論文

【摘要】：蚊媒傳播的瘧疾是一種嚴(yán)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(wèn)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(wèn)題。準(zhǔn)確診斷一直是瘧疾防治工作的一個(gè)重要環(huán)節(jié),顯微鏡檢查作為金標(biāo)準(zhǔn)目前仍是診斷瘧疾的主要手段。但是鏡檢需要專業(yè)的實(shí)驗(yàn)操作技術(shù)和豐富的經(jīng)驗(yàn),如今有經(jīng)驗(yàn)的鏡檢人員越來(lái)越缺乏；PCR檢測(cè)是另一種廣泛使用的方法,盡管其敏感性高,但耗費(fèi)時(shí)間長(zhǎng)且需要特殊儀器、檢測(cè)成本較高。因此迫切需要一種敏感、特異和簡(jiǎn)便的瘧原蟲(chóng)檢測(cè)方法。 目的運(yùn)用環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(Loop-mediated Isothermal Amplification,LAMP)建立一種快速、簡(jiǎn)便、敏感度高的瘧原蟲(chóng)檢測(cè)方法。 方法本研究確定瘧原蟲(chóng)環(huán)子孢子蛋白(CSP)基因?yàn)榘谢?設(shè)計(jì)合成特異性LAMP引物。構(gòu)建間日瘧原蟲(chóng)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(yáng)性對(duì)照。優(yōu)化Mg2+濃度、dNTPs濃度、Bst DNA聚合酶添加量、反應(yīng)溫度、時(shí)間以及設(shè)計(jì)引物缺省試驗(yàn)。評(píng)估優(yōu)化后的LAMP反應(yīng)的特異性和靈敏性。檢測(cè)133份患者血樣,以顯微鏡檢方法為金標(biāo)準(zhǔn),比較LAMP和多重PCR法檢測(cè)間日瘧原蟲(chóng)的敏感性和特異性。初步應(yīng)用LAMP法檢測(cè)斯氏按蚊體內(nèi)鼠約氏瘧原蟲(chóng),對(duì)其特異性和靈敏性進(jìn)行評(píng)估。 結(jié)果建立了采用LAMP技術(shù)快速檢測(cè)人體內(nèi)間日瘧原蟲(chóng)的方法,LAMP法檢測(cè)重組質(zhì)粒DNA (Pv-rDNA)的靈敏度達(dá)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(cè)間日瘧原蟲(chóng)的敏感性為98.53%和97.06%,兩法檢測(cè)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(yáng)性預(yù)測(cè)值和陰性預(yù)測(cè)值分別為88.16%和98.25%,多重PCR的陽(yáng)性預(yù)測(cè)值和陰性預(yù)測(cè)值分別為100%和97.01%。選擇斯氏瘧原蟲(chóng)唾液腺子孢子微線體SPECT2作為靶基因合成LAMP反應(yīng)引物,用該法初步應(yīng)用于斯氏按蚊體內(nèi)鼠約氏瘧原蟲(chóng)的檢測(cè),特異性好,敏感性為能檢測(cè)出在80只陰性斯氏按蚊中有1只約氏瘧原蟲(chóng)陽(yáng)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(chóng)LAMP檢測(cè)方法具有快速簡(jiǎn)便、敏感性高、設(shè)備要求低、成本低廉的特點(diǎn),具有較好的現(xiàn)場(chǎng)應(yīng)用前景；LAMP法初步應(yīng)用于斯氏按蚊體內(nèi)鼠約氏瘧原蟲(chóng)的檢測(cè),特異性和敏感性均較好,為進(jìn)一步研究蚊體內(nèi)瘧原蟲(chóng)子孢子LAMP法檢測(cè)奠定基礎(chǔ)。
[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(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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