越冬白头鹤（Grus monacha）肠道寄生线虫分子检测方法的建立
发布时间：2018-12-14 15:54
【摘要】：寄生虫在野生鸟类中感染相当普遍,在寄生虫的影响下鸟类可能发生疾病,造成生长发育停滞,甚至发生致死性的严重疾病等,因此成为保护生物学研究的热点问题之一。白头鹤(Grus monacha)是一种脆弱的大型迁徙水鸟,种群保护的形势十分严峻,考虑其濒危性在进行寄生虫感染状况调查时大多采取最简单伤害最小的非损伤性取样法取其粪便样品。据以往研究表明,在白头鹤粪便中能检出大量的肠道寄生虫,主要包括球虫,线虫和吸虫等。但是由于传统的形态学鉴别方法的局限性,寄生虫的种类鉴别研究并不透彻,建立白头鹤肠道寄生虫的分子检测方法对寄生虫进行快速、准确的鉴别尤为重要。本研究主要探讨野生白头鹤肠道寄生线虫感染的分子检测方法。于2013年11月至2015年2月期间,收集了在长江中下游地区鄱阳湖、菜子湖和升金湖越冬的白头鹤粪便样本共908份。采用饱和盐水漂浮法进行白头鹤肠道寄生虫形态学鉴定,白头鹤粪便样品中共检测出5类寄生线虫,即毛细线虫(Capillaria spp.)、类圆线虫(Strongyloides spp.)、禽蛔虫(Ascaridia spp.)、毛圆线虫(Trichostrongylus spp.)、钩虫(Ancylostomatidae spp.)等。线虫感染率最高的种类为禽蛔虫(6.9%),其次为毛细线虫(4.6%)、类圆线虫(4.1%)。在有限稀释法对越冬白头鹤粪便样品中寄生虫卵分离的基础上,进行了白头鹤种群肠道寄生虫分子检测方法的探索。通过对毛细线虫单虫卵分别进行液氮/85℃水浴、超声破碎、裂解液消化等三种前处理后进行荧光PCR检测,结果表明单虫卵经裂解液消化后用于荧光PCR检测的效果明显而稳定。所建立的寄生虫卵荧光PCR检测方法敏感性强,可以满足微量模板高检出率的要求。粪便经漂浮和短暂的离心沉淀后即可进行荧光PCR检测。检测过程中镜检步骤的省略使得非专业人员即可操作,减少了工作量,并能够满足快速、批量的寄生虫检测需要。本研究建立的方法中,寄生虫虫卵经一步预处理即可,RT-PCR能够快速准确地检测出阳性样品,显著提高检测效率,且经实验验证检测效率高于镜检处理。本研究借助荧光PCR检测的高度特异性,设计毛细线虫ITS区引物,经特异性实验验证表明与钩虫、毛圆线虫等鹤类常见寄生虫无交叉反应。此方法的探索可为今后毛细线虫的分子检测提供可靠的方法,并为粪便中其他寄生虫的检测提供了一条有效途径。
[Abstract]:Parasites infection is very common in wild birds. Under the influence of parasites, birds may suffer from diseases, resulting in stagnation of growth and development, and even fatal serious diseases, so it has become one of the hot issues in the research of conservation biology. (Grus monacha) is a kind of fragile large migratory waterfowl, and the situation of population protection is very serious. When investigating the parasite infection status, the most simple non-invasive sampling method is used to collect the fecal samples. According to previous studies, a large number of intestinal parasites, including coccidia, nematode and trematode, can be detected in the droppings of white-headed cranes. However, due to the limitations of traditional morphological identification methods, the study of parasite species identification is not thorough. It is very important to establish a molecular detection method to identify parasites quickly and accurately. In this study, molecular detection methods of parasitic nematode infection in the intestinal tract of wild cranes were studied. From November 2013 to February 2015, 908 samples of white-headed cranes were collected from Poyang Lake, Caizi Lake and Shengjin Lake in the middle and lower reaches of the Yangtze River. The morphology of intestinal parasites was identified by saturated saline floating method. Five parasitic nematode species, (Capillaria spp.), nematode, (Strongyloides spp.), roundworm, Ascaris lumbricoides (Ascaridia spp.), were detected in fecal samples of Crane Whitehead Crane. (Trichostrongylus spp.), hookworm (Ancylostomatidae spp.) Etc. The highest infection rate of nematodes was avian roundworm (6.9%), followed by nematode capillaris (4.6%) and nematoid nematode (4.1%). Based on the separation of parasite eggs from the feces of overwintering white-headed cranes by finite dilution method, the molecular detection method of intestinal parasites of white-headed cranes was explored. Three kinds of pre-treatment, such as liquid nitrogen / 85 鈩，
本文编号：2378902
[Abstract]:Parasites infection is very common in wild birds. Under the influence of parasites, birds may suffer from diseases, resulting in stagnation of growth and development, and even fatal serious diseases, so it has become one of the hot issues in the research of conservation biology. (Grus monacha) is a kind of fragile large migratory waterfowl, and the situation of population protection is very serious. When investigating the parasite infection status, the most simple non-invasive sampling method is used to collect the fecal samples. According to previous studies, a large number of intestinal parasites, including coccidia, nematode and trematode, can be detected in the droppings of white-headed cranes. However, due to the limitations of traditional morphological identification methods, the study of parasite species identification is not thorough. It is very important to establish a molecular detection method to identify parasites quickly and accurately. In this study, molecular detection methods of parasitic nematode infection in the intestinal tract of wild cranes were studied. From November 2013 to February 2015, 908 samples of white-headed cranes were collected from Poyang Lake, Caizi Lake and Shengjin Lake in the middle and lower reaches of the Yangtze River. The morphology of intestinal parasites was identified by saturated saline floating method. Five parasitic nematode species, (Capillaria spp.), nematode, (Strongyloides spp.), roundworm, Ascaris lumbricoides (Ascaridia spp.), were detected in fecal samples of Crane Whitehead Crane. (Trichostrongylus spp.), hookworm (Ancylostomatidae spp.) Etc. The highest infection rate of nematodes was avian roundworm (6.9%), followed by nematode capillaris (4.6%) and nematoid nematode (4.1%). Based on the separation of parasite eggs from the feces of overwintering white-headed cranes by finite dilution method, the molecular detection method of intestinal parasites of white-headed cranes was explored. Three kinds of pre-treatment, such as liquid nitrogen / 85 鈩，
本文编号：2378902
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