鹅新城疫病毒RT-LAMP可视化检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.01.003

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (1): 22-31.doi: 10.16431/j.cnki.1671-7236.2018.01.003

鹅新城疫病毒RT-LAMP可视化检测方法的建立

刘文俊¹, 黄运茂¹, 阳佑天², 许丹宁¹, 曹楠¹, 周德荣¹, 田允波¹

1. 仲恺农业工程学院, 广东省水禽健康养殖重点实验室, 广州 510225;
2. 佛山科学技术学院, 佛山 528000

收稿日期:2017-06-13 出版日期:2018-01-20 发布日期:2018-01-20
通讯作者: 田允波 E-mail:tyunbo@126.com
作者简介:刘文俊(1988-),男,湖南衡阳人,博士,讲师,研究方向:动物微生物学与免疫学,E-mail:lwjhero123@126.com
基金资助:
广东省科技计划（2017A020208069）；广东省普通高校青年人才创新项目（20161076）；广东大学生科技创新攀登计划专项（pdjh2016b0243）

Establishment of RT-LAMP Visual Detection Method for Goose Newcastle Disease Virus

LIU Wenjun¹, HUANG Yunmao¹, YANG Youtian², XU Danning¹, CAO Nan¹, ZHOU Derong¹, TIAN Yunbo¹

1. Key Laboratory of Waterfowl Healthy Breeding in Guangdong, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
2. Foshan University, Foshan 528000, China

Received:2017-06-13 Online:2018-01-20 Published:2018-01-20

摘要/Abstract

摘要：

本研究拟建立一种能在基层实时、便捷诊断鹅新城疫病毒（goose Newcastle disease virus，GNDV）的检测方法。根据GenBank中公布的GNDV F基因的高度同源保守序列设计特异性引物，并在反应体系中添加钙黄绿素/氯化锰指示剂，建立可视化RT-LAMP检测方法。结果显示，建立的方法能特异地检测出GNDV及NDV Lasota疫苗株，而小鹅瘟病毒（GPV）、禽流感病毒（AIV）、鸭坦布苏病毒（DTMUV）等其他病毒均为阴性。所建立的GNDV可视化RT-LAMP检测方法对RNA的最低检测限为10 pg，反应过程不需PCR仪等复杂的仪器，50 min即可完成反应，可经肉眼观察反应体系颜色判定结果。该方法特异性强、灵敏度高，且操作安全、简便、快捷，可满足基层筛查GNDV的需求。

关键词: 鹅新城疫病毒(GNDV); 环介导等温扩增(LAMP); 钙黄绿素

Abstract:

This study was aimed to establish a Real-time and easy-to-diagnose method for the detection of goose Newcastle disease virus (GNDV) at the grassroots level. Based on the highly homologous conserved sequence of GNDV F gene published in GenBank, specific primers were designed and the calcein/manganese chloride indicator was added to the reaction system to establish a visual RT-LAMP detection method. The results showed that the detection method could specifically detect GNDV and NDV Lasota vaccine strains, and other viruses such as GPV, AIV and DTMUV were negative. The minimum detection limit of RNA was 10 pg. The reaction procedure did not require complex instruments such as PCR instrument, and the reaction was completed within 50 min. The results were observed by the naked eye. This method was specificity, high sensitivity, and safe operation, simple and fast, could meet the primary screening of GNDV.

Key words: goose Newcastle disease virus (GNDV); loop mediated isothermal amplification (LAMP); calcein

中图分类号:

S858.33

刘文俊, 黄运茂, 阳佑天, 许丹宁, 曹楠, 周德荣, 田允波. 鹅新城疫病毒RT-LAMP可视化检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(1): 22-31.

LIU Wenjun, HUANG Yunmao, YANG Youtian, XU Danning, CAO Nan, ZHOU Derong, TIAN Yunbo. Establishment of RT-LAMP Visual Detection Method for Goose Newcastle Disease Virus[J]. , 2018, 45(1): 22-31.

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鹅新城疫病毒RT-LAMP可视化检测方法的建立

Establishment of RT-LAMP Visual Detection Method for Goose Newcastle Disease Virus

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