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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：2115 下载量：804
| 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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