奶牛附红细胞体的分类鉴定及诊断方法的建立

为了从分子水平上确定奶牛附红细胞体（E．wenyoni）的分类学地位及建立奶牛附红细胞体感染诊断方法。本研究通过利用原核生物16S rRNA基因通用引物，对分离得到的奶牛附红细胞体（广西株，E．wenyoni-GX）进行16SrRNA基因的克隆及测序。并建立系统发育进化树；同时根据测序结果设计诊断引物，建立奶牛附红细胞体感染的PCR诊断方法。结果扩增出长约1．5kbp的奶牛附红细胞体的16SrRNA基因片段；特异性试验和敏感性试验表明。所建立的PCR方法与常见支原体、细菌及原虫元交叉反应，能检测奶牛附红细胞体最低DNA量为0．145fg。通过试验结果分析，建议将奶牛附红细胞体这类血营养菌划归入支原体科、支原体属；同时，所建立的PCR诊断方法是特异、敏感、快速的，可应用于临床检测。

Classification of Eperythrozoon wenyoni and development of a diagnostic assay for E.wenyoni infection

In order to classify E.wenyoni and diagnose E.wenyoni infection, cloning and sequencing of the 16S rRNA gene of E. wenyoni isoloted from Guangxi (E.wenyoni-GX) were performed using universal primer. At the same time, we set up a phylogenetic tree and designed a pair of primer based on the 16S rRNA gene of E.Wenyoni-GX to establish a diagnostic assay for E.wenyoni infection. A 1500 bp frogment of E.wenyoni 16S rRNA gene was amplified. The specific and sensitive tests showed that there was no cross reaction with commonly-seen bacteria, Mycoplasma and Protozoa. The smallest amount of detectable E.wenyoni DNA was 0.145 fg. The results from analysis suggest that the Eperythrozoon and haemobartonella genera should be reclassified into Mycoplasma genus of Mycoplasmatales. This PCR assay is specific, sensitive and quick to perform. It can be used for the clinical diagnosis of E. wenyoni infection..