本文選題：嬰兒利什曼原蟲　切入點(diǎn)：動(dòng)基體小環(huán)DNA　出處：《中國(guó)疾病預(yù)防控制中心》2013年碩士論文

【摘要】：目的 利什曼病是由利什曼原蟲無鞭毛體寄生在包括人類在內(nèi)的哺乳動(dòng)物巨噬細(xì)胞而引起的人獸共患寄生蟲病。在我國(guó)當(dāng)前流行的6省(區(qū))中,有四省為動(dòng)物源型,犬是主要傳染源。對(duì)犬的管理是控制此類型黑熱病的關(guān)鍵。因此,研究有效的快速簡(jiǎn)便適于現(xiàn)場(chǎng)使用的檢測(cè)犬利什曼原蟲感染的方法對(duì)控制該型黑熱病具有重要意義。利什曼原蟲動(dòng)基體DNA (kinetoplast DNA, kDNA)是由10-20個(gè)拷貝的大環(huán)和上萬拷貝的小環(huán)構(gòu)成,小環(huán)序列不僅在種間,而且在種內(nèi),甚至在株內(nèi)均存在高度異質(zhì)性。因此,小環(huán)成為特異敏感檢測(cè)利什曼原蟲感染極好的靶分子。環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(1oop-mediated isothermal amplification, LAMP)具有靈敏度高、操作簡(jiǎn)單快速以及對(duì)儀器設(shè)備依賴性低的特點(diǎn)。本研究的目的是建立起以犬眼結(jié)膜為檢測(cè)樣本,以kDNA小環(huán)為檢測(cè)靶分子的環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù),從而為檢測(cè)我國(guó)犬利什曼原蟲感染提供敏感、特異、快速、簡(jiǎn)便、適合于現(xiàn)場(chǎng)應(yīng)用的檢測(cè)方法。 方法 應(yīng)用來自利什曼原蟲kDNA小環(huán)保守區(qū)的一對(duì)特異性引物KP1/KP2擴(kuò)增我國(guó)利什曼原蟲四川株小環(huán)DNA全長(zhǎng)序列并測(cè)序,對(duì)序列進(jìn)行分析；應(yīng)用獲得的序列在線設(shè)計(jì)多組LAMP引物,依據(jù)文獻(xiàn)設(shè)立初始反應(yīng)體系和擴(kuò)增條件對(duì)每組引物的反應(yīng)性進(jìn)行評(píng)價(jià),選取一組能進(jìn)行敏感、特異擴(kuò)增的引物應(yīng)用于LAMP方法建立。應(yīng)用選取的引物確定影響LAMP擴(kuò)增的關(guān)鍵因素Mg2+和dNTPs最適濃度以優(yōu)化反應(yīng)體系,比較濁度法、SYBR Green法和鈣黃綠素法3種觀察反應(yīng)結(jié)果的方法的效果,對(duì)建立的LAMP法的敏感性和特異性進(jìn)行實(shí)驗(yàn)室評(píng)價(jià)。應(yīng)用建立的LAMP方法檢測(cè)現(xiàn)場(chǎng)樣品(來自動(dòng)物源型內(nèi)臟利什曼病流行區(qū)和非流行區(qū)犬眼結(jié)膜拭子,煮沸后直接作為擴(kuò)增模板),并與骨髓鏡檢和靜脈全血常規(guī)PCR檢測(cè)結(jié)果進(jìn)行比較。 結(jié)果 應(yīng)用KP1/KP2引物從汶川株利什曼原蟲擴(kuò)增出約850bp的動(dòng)基體小環(huán)DNA序列。對(duì)隨機(jī)選取的20個(gè)陽性克隆進(jìn)行測(cè)序,并對(duì)序列進(jìn)行了比對(duì)分析,顯示這些序列可區(qū)分為兩大類,其中以CQ18為代表的15個(gè)序列可歸為一大類,占比75%,為優(yōu)勢(shì)序列,序列長(zhǎng)度為858bp,其余以CQ07為代表的5個(gè)序列可歸為一類,占比25%,序列長(zhǎng)度為854bp,CQ18和CQ07兩者序列的一致性為82.7%。進(jìn)化分析顯示這兩類序列與杜氏利什曼原蟲種團(tuán)的原蟲親緣關(guān)系最近。應(yīng)用CQ18序列進(jìn)行在線引物設(shè)計(jì),得到5組引物,對(duì)5組引物分別進(jìn)行LAMP反應(yīng)評(píng)價(jià),只有第2組引物能引起特異、敏感LAMP反應(yīng)；對(duì)反應(yīng)體系進(jìn)行優(yōu)化,顯示Mg2+最適濃度為8mM, dNTPs最適濃度為1.4mM.應(yīng)用3種方法觀察LAMP反應(yīng)結(jié)果,顯示鈣黃綠素法能顯著區(qū)分陽性和陰性LAMP反應(yīng)結(jié)果。建立的LAMP法的檢出下限為10-7個(gè)原蟲/mL,而常規(guī)PCR采用RV1/RV2和K13A/K13B兩對(duì)引物,檢出下限分別為10-3和10-2個(gè)原蟲/mL。應(yīng)用其他8種不同蟲種(株)的利什曼原蟲kDNA進(jìn)行LAMP反應(yīng),只有來自我國(guó)的3株嬰兒利什曼原蟲蟲株JS5、甘犬和801顯示陽性反應(yīng)。應(yīng)用建立的LAMP法、鏡檢法和常規(guī)PCR法檢測(cè)內(nèi)臟利什曼病流行區(qū)犬樣本,陽性率分別為59.46%(66/111)、7.62%(8/105)和48.1%(53/110),LAMP和PCR陽性檢出率無統(tǒng)計(jì)學(xué)差異(X2=2.83,P0.05)。應(yīng)用PCR和LAMP檢測(cè)內(nèi)臟利什曼病非流行區(qū)犬樣本,分別出現(xiàn)1(1/33)和2(2/32)例假陽性,LAMP和PCR陰性檢出率無統(tǒng)計(jì)學(xué)差異(X2=0.0007,P0.05)。 結(jié)論 基于我國(guó)嬰兒利什曼原蟲四川汶川株動(dòng)基體小環(huán)DNA序列設(shè)計(jì)引物,成功建立了以犬眼結(jié)膜組織為檢測(cè)樣本的檢測(cè)我國(guó)犬利什曼原蟲感染LAMP檢測(cè)方法,與常規(guī)PCR法比較,檢測(cè)的特異性相當(dāng),而檢測(cè)的敏感性更高,且取樣簡(jiǎn)便,反應(yīng)快速,適合在現(xiàn)場(chǎng)應(yīng)用。兩種分子方法檢測(cè)結(jié)果均顯示我國(guó)動(dòng)物源型黑熱病疫區(qū)犬利什曼原蟲感染率相當(dāng)高,應(yīng)加強(qiáng)犬的管理以控制當(dāng)?shù)氐暮跓岵　?br/>[Abstract]:objective
Leishmaniasis is caused by Leishmania amastigotes parasitic in mammals including human macrophages caused by parasitic disease. In 6 provinces of China, the current popular (area), there are four provinces for animal source, dogs are the main source of infection. The dog management is the key to control this type of leishmaniasis. Therefore study on fast, simple and effective method which is suitable for field use for detection of canine Leishmania infection has important significance to control the type of leishmaniasis. Leishmania kinetoplast DNA (kinetoplast DNA kDNA) is a copy from 10-20 copies of the big ring and tens of thousands of small, small series not only among the species and within species, and even within a tree are highly heterogeneous. Therefore, become small molecular target specific and sensitive detection of Leishmania infection. Excellent loop mediated isothermal amplification (1oop-mediated isothermal amplification, LAMP) has the advantages of high sensitivity, simple operation and rapid equipment dependent low. The purpose of this study is to establish a canine conjunctival as samples to kDNA ring for detecting target molecules guide loop mediated isothermal amplification technology, so as to provide sensitive, for the detection of our canine Leishmania infection specific, fast. The detection method is simple, suitable for field application.
Method
A pair of specific primers of KP1/KP2 from Leishmania kDNA small environmental protection area of our country keep amplification of full-length DNA sequence of Sichuan strain of Leishmania ring and DNA sequencing, sequence analysis; multiple sets of LAMP primers obtained online application design based on the literature, the establishment of the initial reaction system and evaluate the response of each primer amplification conditions, selection a group of sensitive primer application specific amplification in LAMP method. Primers were selected to determine the key factors affecting the application of LAMP amplification of Mg2+ and dNTPs concentration in order to optimize the reaction system, comparative turbidity method, SYBR method and Green method of calcein method 3 to observe the reaction results of the laboratory evaluation of effect. The established LAMP method. The sensitivity and specificity of the LAMP method to detect field samples (from animal sources of visceral leishmaniasis endemic and non endemic areas in dogs The eye conjunctival swabs were directly used as the amplification template after boiling, and compared with the results of bone marrow microscopy and total venous blood routine PCR.
Result
Application of KP1/KP2 primers amplified kinetoplast minicircle DNA sequence of about 850bp from Wenchuan. Leishmania spp. sequencing of 20 positive clones were randomly selected, and the sequence was compared and showed that these sequences can be divided into two categories, including 15 sequences represented by CQ18 can be classified as a class, accounting for 75%, as the dominant sequence, sequence length of 858bp, the remaining 5 sequences represented by CQ07 can be classified as a class, accounting for 25%, the length of the sequence of 854bp, CQ18 and CQ07 of the two consensus sequences for phylogenetic analysis of 82.7%. recently showed that these two kinds of sequence and Liz Leishmania species groups from genetic protozoa application of CQ18 series online. Primer design, 5 sets of primers, 5 primers of group were evaluated in LAMP, only second sets of primers can cause specific and sensitive LAMP reaction; the reaction system was optimized, the optimum Mg2+ concentration is 8mM, dNTPs The optimum concentration of 1.4mM. was observed in the LAMP reaction results of application of the 3 methods, show the calcein method can significantly distinguish between positive and negative LAMP results. The detection limits of the LAMP method established for 10-7 protozoa /mL, while conventional PCR and K13A/K13B RV1/RV2 using two pairs of primers, detection limits were 10-3 and 10-2 protozoa /mL. application the other 8 different species (strains) of Leishmania kDNA LAMP reaction, only from the 3 strains of baby Lishman parasite strains JS5, Gansu dogs and 801 showed positive reaction. The LAMP method is used to establish the microscopic method and conventional PCR method to detect canine visceral leishmaniasis endemic area of samples, the positive rate was 59.46% (66/111), 7.62% (8/105) and 48.1% (53/110), LAMP and PCR positive rate showed no significant difference (X2=2.83, P0.05). The application of PCR and LAMP detection of visceral leishmaniasis in non epidemic area of canine samples, there were 1 (1/33) and 2 (2/32) false positive, L The negative detection rates of AMP and PCR were not statistically significant (X2=0.0007, P0.05).
conclusion
Based on Sichuan Wenchuan strains of Leishmania infantum kinetoplast minicircle DNA primers were successfully established to detect canine conjunctival tissue samples for detection of canine Leishmania infection in China LAMP detection method, compared with the conventional PCR method, the specificity of detection, and detection sensitivity is higher, and the sampling is simple, quick response, suitable for application in the field. The results of the two methods of molecular detection showed animal China black fever epidemic areas of canine Leishmania infection rate is very high, we should strengthen the management of dogs to control the local black fever.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R382.22;R3416

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