本文選題：人五毛滴蟲 + 18S�。� 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：人五毛滴蟲為一種機(jī)會(huì)性致病的人獸共患的寄生蟲,多寄生于宿主的盲腸、結(jié)腸,宿主范圍廣泛。近幾年,臨床人感染人五毛滴蟲的病例常有報(bào)道,主要分布在我國(guó)河南、安徽、湖北、廣東等地,而在動(dòng)物糞便樣品檢測(cè)中也發(fā)現(xiàn)了人五毛滴蟲的感染,已檢測(cè)到的有犬、貓、非靈長(zhǎng)類和嚙齒類動(dòng)物感染。為了探索鹿感染人五毛滴蟲的情況并進(jìn)一步評(píng)估其人獸共患潛力,就顯得十分有必要構(gòu)建一個(gè)快速有效的檢測(cè)方法來評(píng)估鹿感染人五毛滴蟲的情況。腸道毛滴蟲的檢測(cè)常用方法包括:直接涂片鏡檢法、染色鏡檢法、普通PCR檢測(cè)法、蟲體培養(yǎng)法、巢氏PCR檢測(cè)法、原位雜交檢測(cè)法等,其中蟲體培養(yǎng)法、直接涂片鏡檢法、蟲體培養(yǎng)法、普通PCR檢測(cè)法和染色鏡檢法精確度不高,原位雜交檢測(cè)法實(shí)驗(yàn)條件要求高且步驟復(fù)雜,而巢式PCR檢測(cè)法常被視為一種快速、便捷、有效的檢測(cè)方法。本試驗(yàn)通過18S r RNA設(shè)計(jì)了特異性的巢式PCR引物,對(duì)鎂離子濃度及退火溫度進(jìn)行了優(yōu)化,確定了最佳鎂離子濃度為2.5 mmol/L,兩輪PCR反應(yīng)的最佳退火溫度均為53℃,在對(duì)巢式PCR檢測(cè)方法的特異性試驗(yàn)中發(fā)現(xiàn)其與柔嫩艾美爾球蟲、伊氏錐蟲、弓形蟲、陰道毛滴蟲、藍(lán)氏賈第蟲、犬心絲蟲基因組DNA等無(wú)交叉反應(yīng),可檢測(cè)靈敏度達(dá)10個(gè)蟲體/ml。采用構(gòu)建的基于18S r RNA基因的巢式PCR檢測(cè)方法對(duì)長(zhǎng)春地區(qū)139份鹿糞便樣品進(jìn)行了檢測(cè),檢測(cè)結(jié)果顯示人五毛滴蟲感染率為21.59%(30/139),巴特里四毛滴蟲感染率為54.68%(76/139)。采用EF1-α基因設(shè)計(jì)了特異性的巢式PCR引物,對(duì)相關(guān)影響因素條件進(jìn)行了優(yōu)化,確定了最佳鎂離子濃度為0.75mmol/L,兩輪PCR反應(yīng)的最佳退火溫度分別為57℃、55℃,在對(duì)巢式PCR檢測(cè)方法的特異性試驗(yàn)中發(fā)現(xiàn)其與柔嫩艾美爾球蟲、伊氏錐蟲、弓形蟲、陰道毛滴蟲、藍(lán)氏賈第蟲、犬心絲蟲基因組DNA等無(wú)交叉反應(yīng),可檢測(cè)靈敏度達(dá)10個(gè)蟲體/ml。應(yīng)用基于EF1-α基因構(gòu)建的巢式PCR檢測(cè)方法,檢測(cè)對(duì)長(zhǎng)春地區(qū)139份鹿糞便樣品,檢測(cè)結(jié)果顯示人五毛滴蟲感染率為21.59%(30/139)。通過對(duì)陽(yáng)性樣品的序列進(jìn)行分析,結(jié)果顯示,檢測(cè)到的人五毛滴蟲陽(yáng)性樣品的18S r RNA基因序列突變位點(diǎn)主要集中在三個(gè)部位,即第1034個(gè)堿基存在著C/T的差異,第1051-1053的堿基存在著TGC/TGT/GAT的差異,第1144-1165的堿基存在著復(fù)雜的多樣性;檢測(cè)到的人五毛滴蟲陽(yáng)性樣品的EF-1α基因序列未表現(xiàn)出多態(tài)性。綜上所述,基于人五毛滴蟲EF-1α基因的巢式PCR檢測(cè)方法更適合檢測(cè)人五毛滴蟲的感染情況,而基于18S r RNA基因的巢式PCR檢測(cè)方法更適合用于人五毛滴蟲基因亞型的鑒定,在臨床檢測(cè)過程中,可先用基于EF-1α基因的巢式PCR檢測(cè)方法進(jìn)行檢測(cè),將檢測(cè)到的陽(yáng)性樣品再采用基于18S r RNA基因的巢式PCR檢測(cè)方法進(jìn)行基因分型等的研究。本試驗(yàn)成功的構(gòu)建了基于特異性診斷基因的診斷方法,為流行病學(xué)的研究提供了新的手段。
[Abstract]:Trichomonas hominis is an opportunistic zoonotic parasite that parasitizes the cecum and colon of the host and has a wide range of hosts. In recent years, clinical cases of human trichomonas infection have often been reported, mainly distributed in Henan, Anhui, Hubei, Guangdong and other places in China, and human trichomonas infection has also been found in animal fecal samples. Dogs and cats have been detected. Infection in non-primates and rodents. In order to explore the infection of Trichomonas hominis in deer and further evaluate its zoonotic potential, it is necessary to construct a rapid and effective method to evaluate the infection of Trichomonas hominis in deer. The common methods for detection of trichomonas intestinal trichomonas include direct smear microscopy, staining microscopy, common PCR detection, body culture, nest PCR detection, in situ hybridization, and so on. The methods of insect culture, common PCR and staining microscopy are not accurate, and the in situ hybridization method requires high experimental conditions and the steps are complicated. However, nested PCR detection method is often regarded as a fast, convenient and effective detection method. In this experiment, a specific nested PCR primer was designed by 18s rRNA. The magnesium ion concentration and annealing temperature were optimized. The optimum magnesium ion concentration was 2.5 mmol / L, and the optimal annealing temperature for both rounds of PCR reaction was 53 鈩，

本文編號(hào)：2019772