牛附红细胞体LAMP检测方法的建立

Development of LAMP Assay for E.wenyoni

为建立一种快捷、灵敏的牛附红细胞体检测方法,本研究根据GenBank上发表的16S rRNA基因(登录号:AF016546)序列设计合成2对LAMP特异引物,建立了检测牛附红细胞体环介导等温扩增(LAMP)方法,并优化了LAMP的各反应条件,进行了敏感性和特异性试验。LAMP扩增产物经电泳、显色鉴定。结果显示,LAMP方法扩增牛附红细胞体产物呈特征性梯状条带,显色反应呈现绿色荧光;敏感性检测最低浓度为25.6 fg/μL;特异性试验结果显示,牛附红细胞体检测管显色后呈阳性,而猪附红细胞体、牛新孢子虫、弓形虫及牛瑟氏泰勒虫等对照组均呈阴性,说明本研究建立的LAMP方法具有灵敏、特异、快速等优点,适合于牛附红细胞体的检测。

In order to establish a rapid and sensitive method to detect E.wenyoni,two pairs of LAMP specific primers were designed and synthesized according to the sequence of 16S rRNA gene（AF016546） published in GenBank;loop-mediated amplification（LAMP） for detecting E.wenyoni was constructed and the reaction conditions of LAMP were optimized;sensitivity and specificity tests were carried out.LAMP results were judged by colorable reaction,electrophoretic analysis.The results showed that the LAMP product in E.wenyoni tube were confirmed by gel electrophoresis and a characteristic DNA ladder was observed.After adding SYBR Green Ⅰ to the reaction tube,green fluorescence was observed with E.wenyoni tube.The detection limit of LAMP assay was 25.6 fg/μL DNA.The specific test proved that in colorable reaction the detection tube of E.wenyoni was positive,while Eperythrozoon suis,bovine Neospora,Toxoplasma gondii,Theileria sergenti and as the comparisons were all negative.This research demonstrated that LAMP was a sensitive,specific and rapid method which was suitable for detection of E.wenyoni.