贝氏贝诺孢子虫遗传进化分析及巢氏PCR检测方法的建立

发布时间：2018-08-08 13:04

【摘要】：贝氏贝诺孢子虫（Besnoitia besnoiti）主要寄生于牛的眼、皮肤和生殖系统等部位，引起牛贝诺孢子虫病（Besnoitiosis）。牛感染后会出现消瘦、贫血、水肿及消化功能障碍，导致母牛流产、产奶量下降、公牛精液质量下降，严重感染时可引起死亡，给养牛业带来重大的经济损失。目前对贝诺孢子虫的研究主要集中在贝诺孢子虫病的流行病学、诊断和综合防治等传统寄生虫学方面，在分子水平上仅对核糖体内转录间隔区1（internal transcribed spacer ITS1），进行了研究，对线粒体全基因组的研究还未见报道。因此，本研究的第一部分内容是利用分子生物学的方法，扩增贝氏贝诺孢子虫的线粒体全基因组和贝氏贝诺孢子虫中国株的ITS-1序列，对获得的整个线粒体基因组序列和ITS-1进行测序和序列分析，并基于线粒体基因组蛋白质编码基因序列和核糖体rDNA的ITS-1构建系统发生树，探讨贝氏贝诺孢子虫与孢子虫科其他原虫的进化关系，确定其在原虫中的分类地位。结果，获得的贝氏贝诺孢子虫线粒体全基因组由2个蛋白质编码基因和7个tRNA基因组成，大小为3679bp。整个线粒体基因组中核酸组成为A=29.14%，C=19.30%，G=18.48%，T=33.08%，A+T占62.22%，G+C占37.78%，基因组碱基组成明显偏好碱基A和T。以串联的线粒体2个蛋白质编码基因为基因标记，采用BI、MP和ML法构建系统发生树的结果相似，进化树分为二个大的分支，叶足亚纲和球虫亚纲各为一支。在球虫亚纲中，真球虫目和梨形虫目各为一独立分支。在真球虫目中，艾美耳亚目和血孢子亚目各为一个大的分支。在艾美耳亚目中，艾美耳科为一个大的分支，其他科共同位于一个分支上。而贝氏贝诺孢子虫与刚地弓形虫、新孢子虫和哈蒙球虫在一个姐妹分支上，是亲缘关系最近的虫种。此结果与传统形态学分类一致，，说明线粒体全基因序列是研究寄生虫分子种系发生的良好基因标记。以所获得的ITS-1rDNA为基因标记，采用MP、ML和BI三种方法构建系统发生树的结果相似，贝氏贝诺孢子虫与刚地弓形虫、新孢子虫和哈蒙球虫在一个姐妹分支上。此结果与以线粒体全基因序列为标记基因构建的进化树结果相似，同时贝氏贝诺孢子虫中国株和美国、德国、澳大利亚、以色列等国家检出的病原一致。贝氏贝诺孢子虫病诊断方法的研究一直还停留在最原始的临床观察诊断方面，但是对感染贝氏贝诺孢子虫的病牛来说，在感染初期，病牛临床表现不明显，出现临床症状的时候已经到了晚期，只能选择淘汰，给畜牧业造成重大的经济损失。因此本研究的第二部分内容是在分子水平上，建立一种快速、准确的诊断方法，对疾病进行早期诊断，以减少经济损失。根据已发表的贝诺孢子虫rDNA序列，设计贝氏贝诺孢子虫rDNAITS巢氏引物。提取病牛含有贝氏贝诺孢子虫血液的基因组总DNA，进行巢氏PCR扩增和测序，建立一种快速、特异、敏感的巢氏PCR诊断方法，并应用该方法对黑龙江省部分地区196份奶牛血液样品进行感染情况检查。结果表明，建立的贝诺孢子虫巢式PCR方法具有敏感、特异的优点；临床检测出阳性血样3份，阳性率为1.53%。本研究首次获得了贝氏贝诺孢子虫的线粒体全基因组序列，并对其特征进行了分析，丰富了原虫线粒体全基因组资料，基于线粒体基因组蛋白编码序列和ITS-1序列构建的系统发生树从分子水平上明确了贝氏贝诺孢子虫及其他原虫的进化关系，为其它寄生原虫群体遗传学、系统发生学的研究奠定了基础。首次建立了奶牛贝诺孢子虫巢氏PCR的诊断方法，并对黑龙江省部分地区奶牛感染情况进行初步调查，为该病进一步的防控提供可靠依据。
[Abstract]:Besnoitia besnoiti mainly parasitic on the eyes, skin, and reproductive systems of cattle, causing bovine bainosporidiosis (Besnoitiosis). After cattle infection, emaciation, anemia, edema and digestive dysfunction may result in abortion, lower milk production and the quality of cow semen, death and supply can be caused in severe infection. The cattle industry has brought great economic losses.
At present, the study of Sporozoa Benno mainly focuses on the epidemiology, diagnosis and comprehensive prevention and control of the disease of Benno Sporozoa. At the molecular level, only the transcriptional spacer zone 1 (internal transcribed spacer ITS1) of ribose is studied. The study of the whole genome of the nematome has not been reported. The first part is to amplify the ITS-1 sequence of the whole genome of the mitochondria and the Chinese strain of the Chinese strain of baineno beetle by molecular biology. The sequence and sequence analysis of the whole mitochondrial genome sequence and ITS-1 are carried out, based on the sequence of the gene encoding gene of the mitochondrial genome and the ribosome R. DNA's ITS-1 constructs a phylogenetic tree to explore the evolutionary relationship between the Sporozoa baino spore and other protozoa, and determine its classification status in the protozoa. Results, the complete genome of the mitochondrial genome of the Sporozoa baineno sporozoite consists of 2 protein encoding genes and 7 tRNA genes, and the size is nucleic acid in the whole mitochondrial genome of 3679bp.. The composition of A=29.14%, C=19.30%, G=18.48%, T=33.08%, A+T accounted for 62.22%, G+C accounted for 37.78%. The genome base composition obviously preferred the base A and T. in series of mitochondrial 2 protein coding genes as gene markers. The results were similar with BI, MP and ML method for constructing phylogeny tree, and the intake tree was divided into two large branches, leaf subclass and coccidiosis. In the subclass of coccidia, the Eimeria and the suborder of the genus pyriform are an independent branch. In the order Eimeria, the amieriera and the suborder of the blood spores are a large branch. In the amieriera, eimerid is a large branch and the other families are located on a branch. In a sister branch of a sister branch, the parasite and the Harmon coccidia are the closest species of the relationship. This result is consistent with the traditional morphological classification, indicating that the mitochondrial whole gene sequence is a good genetic marker for the study of the parasite molecular phylogeny. The ITS-1rDNA was used as the gene marker to construct the phylogeny tree with three methods, MP, ML and BI. The results were similar. The results were similar to those of the phylogenetic tree constructed with the sequence of mitochondrial gene sequences. The results were similar to those in the United States, Germany, Australia and Israel.
The study of the diagnosis method of bainobo sporozoite has always stayed in the most primitive clinical observation, but for the infected cattle infected with bainobella spore, the clinical symptoms of the infected cattle were not obvious in the early stage of infection. When the symptoms appeared, they had come to the late stage and could only choose and eliminate them, causing major economic damage to the animal husbandry. Therefore, the second part of this study is to establish a rapid and accurate diagnostic method at the molecular level, to diagnose the disease early in order to reduce the economic loss. According to the published rDNA sequence of the sporozoite of Benno, the rDNAITS nesting primers of the Sporozoa bainite were designed. The extraction of the genes of the infected cattle containing the blood of the Benno Sporozoa was extracted. Group DNA, nest PCR amplification and sequencing, a rapid, specific and sensitive nesting PCR diagnosis method was established, and the method was used to detect the infection of blood samples of 196 dairy cows in parts of Heilongjiang province. The results showed that the established nested PCR recipe was sensitive and specific, and the positive blood was detected in clinical. 3 samples with a positive rate of 1.53%.
In this study, the whole genome sequence of the Sporozoa baineno Sporozoa was first obtained, and its characteristics were analyzed. The whole genome of the protozoa was enriched. The phylogenetic tree based on the sequence of mitochondrial genome protein and the sequence of ITS-1 sequence clearly defined the progress of the baineno bereno Sporozoa and other protozoa. The relationship was the basis for the study of other parasitic protozoa population genetics and phylogenetic studies. The diagnosis of nesting PCR of the dairy cow Benno Sporozoa was established for the first time, and the infection of dairy cows in some areas of Heilongjiang province was preliminarily investigated to provide a reliable basis for the further prevention and control of the disease.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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