Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene
Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene
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DOI：
10.1590/s0100-736x2012000800014
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发表时间：
2012-08-01
期刊：
影响因子：
--
通讯作者：
Xu, Zhiwen
中科院分区：
文献类型：
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作者：
Ji, Hongwei;Li, Haitao;Xu, Zhiwen
季慧文, 李宏涛, 朱丽, 张慧, 王勇, 左泽昌, 郭文正. & 徐志伟2012. 开发和评估基于 dsbE 样基因的环介导等温扩增 (LAMP) 测定法，用于快速检测胸膜肺炎放线杆菌。巴西兽医协会 32(8): 757-760。四川农业大学兽医学院四川省动物生物技术重点实验室，四川雅安625014电子邮件：abtcxzw@126.com本文报告了用于快速、特异性检测胸膜肺炎放线杆菌（A. pleuropneumoniae）的环介导等温扩增测定（LAMP）的开发和验证。设计了一组源自胸膜肺炎放线菌 dsbE 样基因的 6 个引物，并使用 9 种胸膜肺炎放线菌参考/现场菌株、132 种临床分离株和 9 种其他病原体验证了测定方法。结果表明，所有胸膜肺炎放线菌菌株和标本经LAMP在63℃、60分钟下均呈阳性反应，而其他非放线菌未观察到交叉反应。胸膜肺炎包括副猪嗜血杆菌、大肠杆菌、多杀性巴氏杆菌、支气管败血博德特氏菌、猪链球菌、肠沙门氏菌、葡萄球菌、猪繁殖与呼吸综合征病毒（PRRSV）和伪狂犬病病毒。常规PCR的检测限为每管102 CFU，而LAMP的检测限为每管5个拷贝。因此，LAMP的敏感性高于PCR。此外，LAMP 检测提供了一种快速而简单的胸膜肺炎放线菌检测方法，由于操作简便且仅需要常规水浴或加热块进行反应，因此适用于实验室诊断和笔侧检测。
Ji H. W., Li H. T., Zhu L., Zhang H., Wang Y., Zuo Z.C., Guo W.Z. & Xu Z.W. 2012. Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene. Pesquisa Veterinaria Brasileira 32(8): 757-760. Key Laboratory of Animal Biotechnology, Center of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, Sichuan, 625014, P. R. China. E-mail: abtcxzw@126.comThis paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A. pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63 C for 60 min and no cross-reactivity were observed from other non-A. pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 102 CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.