鸽圆环病毒可视化LAMP检测方法的建立与应用  

Establishment and application of a visual LAMP method for pigeon circovirus detection

作　　者：赵谜 徐飞飞 郑丹阳 赵治钤 程瑞 熊超 王军 穆杨[1] ZHAO Mi;XU Feifei;ZHENG Danyang;ZHAO Zhiqian;CHENG Rui;XIONG Chao;WANG Jun;MU Yang(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Kaige Ronghui Biotechnology(Xi'an)Co.,LTD.,Xi'an 710117,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨陵712100 [2]凯鸽融汇生物科技(西安)有限公司,陕西西安710117

出　　处：《中国兽医科学》2025年第1期39-47,共9页Chinese Veterinary Science

基　　金：凯鸽融汇生物科技(西安)有限公司横向课题(TG20240007)。

摘　　要：以鸽圆环病毒(PiCV)Rep基因高度保守区为靶标设计环介导等温扩增(LAMP)引物,以甲酚红为指示剂,经条件优化和特异性、敏感性评价等,建立了PiCV的可视化LAMP检测方法,并采用建立的PiCV可视化LAMP方法和常规PCR方法分别对35份鸽临床样品进行PiCV检测并对检测结果进行对比。采用建立的方法在64℃反应60 min后再85℃作用10 min,即可通过目测反应液颜色变化判定检测结果。阳性反应管的反应液由紫红色变为橙色,阴性管反应液颜色不变。采用建立的方法检测鸽腺病毒、鸽疱疹病毒、鸽痘病毒和鸽Ⅰ型副黏病毒等均未出现交叉反应;该方法的最低检测限为17.4 copies/μL重组质粒,表明该方法的灵敏度是常规PCR方法的100倍。采用建立的方法检出PiCV阳性样品16份,常规PCR方法检出阳性样品15份,说明本研究中建立的PiCV可视化LAMP方法可应用于临床检测,为方便快捷地诊断鸽圆环病毒病提供了技术支持。Primers were designed targeting the highly conserved region of pigeon circovirus(PiCV)Rep gene,cresol red was used as an indicator,and a visual loop-mediated isothermal amplification(LAMP)method was established for PiCV detection after condition optimization and specificity and sensitivity evaluation.Then thirty-five clinical samples of pigeons were detected using the established method and the conventional PCR method.It showed that the detection result could be determined by a visual color change of the reaction liquid after reacting at 64℃ for 60 min and then at 85℃ for 10 min.The reaction liquid of the positive reaction tube changed from purple-red to orange,and the color of the negative reaction tube remained unchanged.No cross-reactivity was found in the detection of pigeon adenovirus,pigeon herpesvirus,pigeonpox virus,and pigeon paramyxovirusⅠby the established method.The lowest detection limit was 17.4 copies/μL recombinant plasmid,indicating its sensitivity was 100 times that of the conventional PCR method.Sixteen positive samples were detected by the visual LAMP method,while 15were detected by the conventional PCR method.It indicates that the visual LAMP method established in this study can be applied to clinical sample detection,which provides technical support for convenient and rapid diagnosis of pigeon circovirus infection.

关 键 词：鸽圆环病毒 可视化 环介导等温扩增 甲酚红 建立 

分 类 号：S852.659.2[农业科学—基础兽医学]