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猪肉及其制品中弓形虫RT-LAMP快速检测技术研究

发表时间:2014-06-15  浏览量:1917  下载量:736
全部作者: 曲道峰,韩剑众,杜爱芳,刘晓宁
作者单位: 浙江工商大学食品与生物工程学院;浙江大学动物科学学院;余杭区农业局物流中心检疫站
摘 要: 为提高猪肉及其制品中弓形虫(Toxoplasma gondii)的检出率,研究采用逆转录环介导等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP),建立一种快速、灵敏、特异的检测弓形虫的方法。根据GenBank中弓形虫18S rRNA序列,设计6条RT-LAMP引物,优化反应体系条件,在Bst DNA聚合酶的作用下,65℃反应60 min,加入SYBR Green I后,进行肉眼观察及凝胶电泳分析。结果表明:该RT-LAMP检测体系特异性强,与隐孢子虫RNA等不发生交叉反应;敏感性高,为一般RT-PCR方法的10倍。该方法具有简单、快速、低成本的特点,可用于猪肉及其制品弓形虫的快速检测。
关 键 词: 畜牧、兽医科学其他学科;弓形虫;逆转录环介导等温扩增技术;18S rRNA;检测;猪肉及其制品
Title: Study on the RT-LAMP as a diagnostic tool of Toxoplasma gondii in pork and its products
Author: QU Daofeng, HAN Jianzhong, DU Aifang, LIU Xiaoning
Organization: School of Food Science and Biotechnology, Zhejiang Gongshang University; College of Animal Sciences, Zhejiang University; Logistics Center Quarantine Station of Yuhang District Agricultural Bureau
Abstract: In this study, a fast, sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of Toxoplasma gondii in pork and its products was developed. We used a conserved sequence of 18S rRNA of T. gondii in GenBank to design primers for RT-LAMP test. The amplication was able to finish in 60 min under Bst DNA polymerase at 65℃ by employing a set of six primers. With the addition of the SYBR Green I, we analysized gel electrophoresis results. The assay showed 10 times higher sensitivity than RT-PCR using T. gondii RNA as template. The RT-LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. As RT-LAMP requires very basic instruments and the results can be obtained by visual observation, this technique provides a simple and reliable tool for inspecting food which are T. gondii-contaminated.
Key words: other subjects of animal husbandry and veterinary medicine science; Toxoplasma gondii; reverse transcription loop-mediated isothermal amplification; 18S rRNA; detection; pork and its products
发表期数: 2014年6月第11期
引用格式: 曲道峰,韩剑众,杜爱芳,等. 猪肉及其制品中弓形虫RT-LAMP快速检测技术研究[J]. 中国科技论文在线精品论文,2014,7(11):1112-1118.
 
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