鹿感染人五毛滴虫巢式PCR检测方法的建立与应用

发布时间：2018-06-15 00:49

  本文选题：人五毛滴虫 + 18S　； 参考：《吉林大学》2015年硕士论文

【摘要】：人五毛滴虫为一种机会性致病的人兽共患的寄生虫,多寄生于宿主的盲肠、结肠,宿主范围广泛。近几年,临床人感染人五毛滴虫的病例常有报道,主要分布在我国河南、安徽、湖北、广东等地,而在动物粪便样品检测中也发现了人五毛滴虫的感染,已检测到的有犬、猫、非灵长类和啮齿类动物感染。为了探索鹿感染人五毛滴虫的情况并进一步评估其人兽共患潜力,就显得十分有必要构建一个快速有效的检测方法来评估鹿感染人五毛滴虫的情况。肠道毛滴虫的检测常用方法包括:直接涂片镜检法、染色镜检法、普通PCR检测法、虫体培养法、巢氏PCR检测法、原位杂交检测法等,其中虫体培养法、直接涂片镜检法、虫体培养法、普通PCR检测法和染色镜检法精确度不高,原位杂交检测法实验条件要求高且步骤复杂,而巢式PCR检测法常被视为一种快速、便捷、有效的检测方法。本试验通过18S r RNA设计了特异性的巢式PCR引物,对镁离子浓度及退火温度进行了优化,确定了最佳镁离子浓度为2.5 mmol/L,两轮PCR反应的最佳退火温度均为53℃,在对巢式PCR检测方法的特异性试验中发现其与柔嫩艾美尔球虫、伊氏锥虫、弓形虫、阴道毛滴虫、蓝氏贾第虫、犬心丝虫基因组DNA等无交叉反应,可检测灵敏度达10个虫体/ml。采用构建的基于18S r RNA基因的巢式PCR检测方法对长春地区139份鹿粪便样品进行了检测,检测结果显示人五毛滴虫感染率为21.59%(30/139),巴特里四毛滴虫感染率为54.68%(76/139)。采用EF1-α基因设计了特异性的巢式PCR引物,对相关影响因素条件进行了优化,确定了最佳镁离子浓度为0.75mmol/L,两轮PCR反应的最佳退火温度分别为57℃、55℃,在对巢式PCR检测方法的特异性试验中发现其与柔嫩艾美尔球虫、伊氏锥虫、弓形虫、阴道毛滴虫、蓝氏贾第虫、犬心丝虫基因组DNA等无交叉反应,可检测灵敏度达10个虫体/ml。应用基于EF1-α基因构建的巢式PCR检测方法,检测对长春地区139份鹿粪便样品,检测结果显示人五毛滴虫感染率为21.59%(30/139)。通过对阳性样品的序列进行分析,结果显示,检测到的人五毛滴虫阳性样品的18S r RNA基因序列突变位点主要集中在三个部位,即第1034个碱基存在着C/T的差异,第1051-1053的碱基存在着TGC/TGT/GAT的差异,第1144-1165的碱基存在着复杂的多样性;检测到的人五毛滴虫阳性样品的EF-1α基因序列未表现出多态性。综上所述,基于人五毛滴虫EF-1α基因的巢式PCR检测方法更适合检测人五毛滴虫的感染情况,而基于18S r RNA基因的巢式PCR检测方法更适合用于人五毛滴虫基因亚型的鉴定,在临床检测过程中,可先用基于EF-1α基因的巢式PCR检测方法进行检测,将检测到的阳性样品再采用基于18S r RNA基因的巢式PCR检测方法进行基因分型等的研究。本试验成功的构建了基于特异性诊断基因的诊断方法,为流行病学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，

本文编号：2019772