摘要
为建立一种快捷、灵敏的牛新孢子虫病检测方法,根据GenBank上登录的新孢子虫NcSAG1基因(AF132217)序列设计合成了2对环介导等温扩增(LAMP)特异引物,建立了检测牛新孢子虫病的LAMP技术,并优化了LAMP的各反应条件,进行了敏感性和特异性试验。结果显示,LAMP方法扩增牛新孢子虫的电泳产物呈特征性梯状条带,显色反应呈现绿色荧光,酶切鉴定出现目的条带,LAMP方法检测的敏感性为5×105copies/μL;特异性试验显示,新孢子虫检测管显色后呈阳性,而瑟氏泰勒虫、弓形虫和附红细胞体等对照组均呈阴性。表明,本研究建立的LAMP方法具有灵敏、特异、快速等优点,适合于牛新孢子虫病的检测。
In order to establish a quick and sensitive method to detect bovine neosporaosis, two specific primer pairs for loop-mediated isothermal amplification（LAMP） were designed and synthesized according to the sequence of Neospora NcSAG1 gene（AF132217） available in GenBank,then a LAMP assay for detecting bovine neosporaosis was constructed and the reaction conditions of LAMP were optimized, and sensitivity and specificity tests were carried out. The results showed that the products amplified by LAMP from bovine Neospora were distinctive ladder-like bands in agarose gel electrophoresis,and the green fluorescence in colorable reaction and that the objective bands occurred by restriction enzyme reaction. The detecting sensitivity was 5 ×10^5 copies/μL. The specificity test proved that the detection tube of Neospora was positive in colorable reaction,while Theileria sergenti, Toxoplasma gondii and Eperythrozoon as the comparisons were all negative. This study demonstrated that LAMP was a sensitive, specific and rapid method which was suitable for the detection of bovine neosporaosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第12期1084-1088,共5页
Chinese Veterinary Science
