鼠痘病毒环介导等温扩增可视化检测方法的建立

doi:10.3969/j.issn.1674-5817.2018.05.004

实验动物与比较医学 ›› 2018, Vol. 38 ›› Issue (5): 343-349.DOI: 10.3969/j.issn.1674-5817.2018.05.004

鼠痘病毒环介导等温扩增可视化检测方法的建立

周洁, 陶凌云, 赵丽娟, 胡建华, 高诚

上海实验动物研究中心,上海 201203

收稿日期:2018-06-14 出版日期:2018-10-25 发布日期:2018-10-25
作者简介:周洁(1978-),女,博士,研究员,主要从事动物病毒分子生物学及免疫学方面的研究。E-mail:zhoujie0526@163.com
基金资助:
上海市科委科技创新行动计划(15140900600);上海市科委研发平台专项(15DZ2292400)

Visualized Detection of Ectromelia Virus by Loop-Mediated Isothermal Amplification Assay

ZHOU Jie, TAO Ling-yun, ZHAO Li-juan, HU Jian-hua, GAO Cheng

Shanghai Laboratory Animal Research Center,Shanghai 201203,China

Received:2018-06-14 Online:2018-10-25 Published:2018-10-25

摘要/Abstract

摘要： 目的应用环介导等温扩增(LAMP)技术建立一种鼠痘病毒(ECTV)的可视化快速检测方法。方法通过序列分析选择3段ECTV序列保守区域设计了9套LAMP引物,利用LAMP Real Time Turbidimeter LA-320C仪监测反应进程并筛选最佳引物组合,优化反应条件,建立了对ECTV核酸进行特异性扩增的LAMP 检测方法,并可通过加入目测荧光检测试剂肉眼判断结果。对所建立的方法进行了敏感性、特异性评估,并对人工感染样品进行了检测。结果该方法在63 ℃恒温下作用60 min,ECTV DNA获得了高效特异性扩增,与其余常见小鼠病毒无交叉反应;最低检出量为530 fg/µL,是常规PCR的10³倍,加入目测荧光染料判断结果与Real Time Turbidimeter LA-320 仪监测结果一致。结论建立的ECTV LAMP 检测方法具有快速、特异、灵敏和操作简单的特点,或具有良好应用前景。

关键词: 鼠痘病毒(ECTV), 环介导等温扩增技术(LAMP), 实时, 快速检测

Abstract: Objective To established a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of ectromelia virus (ECTV). Methods According to the published GenBank sequences (NC_004105.1), 9 pairs of primers were designed targeting the conserved region of ECTV. The amplification was detected with LAMP Real Time Turbidimeter LA-302, and the visudlized detection method of ECTV was established. Meanwhile, the amplified products were colored by fluorescence detection reagent after completion of the reaction, so that the amplification could be detected with naked eyes. Then, methodological evaluation of the LAMP was tested, and the samples were tested for artificial infection. Results The method of LAMP shows a highly efficient amplification for ECTV viral target gene which performed at 63 ℃ for 60 min by the LAMP Real Time Turbidimeter LA-302. The detection limit was 530 fg/µL, was 10³ times higher than that of PCR, and no cross-reaction with other RNA and DNA of viruses from mice. The results of ECTV LAMP reaction visualized the tube directly with naked eyes by the addition of fluorescence detection reagent are consistent with the results detected by Tubidimeter real-time. Conclusion The established LAMP detection method for ECTV is rapid, specificity, high sensitivity, and be easy of operation under simple conditions, which may be suitable for rapid detection of ECTV.

Key words: Ectromelia virus (ECTV), Loop-mediated isothermal amplification (LAMP), Real time, Rapid detection

中图分类号:

Q95-33

周洁, 陶凌云, 赵丽娟, 胡建华, 高诚. 鼠痘病毒环介导等温扩增可视化检测方法的建立[J]. 实验动物与比较医学, 2018, 38(5): 343-349.

ZHOU Jie, TAO Ling-yun, ZHAO Li-juan, HU Jian-hua, GAO Cheng. Visualized Detection of Ectromelia Virus by Loop-Mediated Isothermal Amplification Assay[J]. Laboratory Animal and Comparative Medicine, 2018, 38(5): 343-349.

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鼠痘病毒环介导等温扩增可视化检测方法的建立

Visualized Detection of Ectromelia Virus by Loop-Mediated Isothermal Amplification Assay

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