摘要
【目的】建立特异性好、灵敏度高的非洲猪瘟病毒(African swine fever virus,ASFV)环介导等温扩增(Loop-Mediated Isothermal Amplification,LAMP)快速检测方法,为非洲猪瘟的实时实地快速检测提供参考。【方法】通过对非洲猪瘟病毒多种基因型66个毒株的E165R基因多序列比对,选取相对保守区域设计LAMP引物,并对反应条件及反应体系进行优化。在获得的最优反应条件及体系下考察方法的检测特异性和灵敏性。【结果】选取的LAMP检测靶标235 bp(29~264 bp)在不同基因型ASFV毒株中保守性较好,序列相似度达94.46%~100%;LAMP反应的最佳温度为64℃、时间为35 min、Mg 2+浓度为8 mmol/L、内外引物比为8︰1、Bst DNA聚合酶量为16 U。建立的ASFV环介导等温扩增检测方法最低检测限为50 copies/μL,较常规PCR检测提高1个数量级,且35 min即可完成扩增反应。LAMP检测方法与猪圆环病毒2型(PCV2)、伪狂犬病毒(PRV)、猪繁殖与呼吸道综合症病毒(PRRSV)和猪瘟病毒(CSFV)4种临床症状类似的猪病毒感染性疫病没有交叉反应。【结论】基于该新检测靶点的ASFV LAMP检测具有良好的特异性,可成功区分与ASFV感染临床症状相似的猪感染性疾病,且灵敏度优于常规PCR检测方法,方法简单、检测成本低,可实现ASFV的特异、灵敏、快速检测。
【Objective】The loop-mediated isothermal amplification(LAMP)rapid detection method with good specificity and higher sensitivity to African swine fever virus(ASFV)is established to provide a reference for real-time and on-site rapid detection of ASFV.【Method】The LAMP primers for relative conservation segment are designed by multiple sequence alignment of E165R gene from 66 ASFV strains with different genetypes and the interaction condition and system are optimized.The specificity and sensitivity of the LAMP rapid detection method are investigated under the optimal reaction condition and system.【Result】The sequence similarity of the selected LAMP detection target 235 bp(29~264 bp)with a good conservation among different genotypes of ASFV strains reaches 94.46%~100%.The optimal temperature,time,Mg 2+concentration,inner/outer primer ratio and Bst DNA polymerase amount for LAMP reaction are 64℃,35 min,8 m mol/L,8:1 and 16 U respectively.The minimum detection limit of the established loop-mediated isothermal amplification(LAMP)detection method is 50 copies/μL,which is one order of magnitude higher than the convention PCR detection method.The established loop-mediated isothermal amplification(LAMP)detection method can complete amplification reaction within 35 min.The established LAMP detection method has no cross reaction with 4 pig viral infectious diseases(PCV2,PRV,PRRSV and CSFV)with similar clinical symptoms.【Conclusion】The established LAMP detection method with a good specificity based on a new detection target of ASFV can differentiate between ASFV and other pig infectious diseases with the similar clinical symptoms successfully.The established LAMP detection method with the advantages of good specificity,higher sensitivity(compared with PCR),easy operation and low cost realizes the specific,sensitive and rapid detection of ASFV.
作者
林涛
陈珺煜
王成龙
李芹
张怀东
刘峰
LIN Tao;CHEN Junyu;WANG Chenglong;LI Qin;ZHANG Huaidong;LIU Feng(College of Life Sciences,Fujian Normal University,Fuzhou,Fujian 350117;National and Local Joint Engineering Research Center for Industrial Microbial Fermentation Technology,Fuzhou,Fujian 350117,China)
出处
《贵州农业科学》
CAS
2023年第3期82-88,共7页
Guizhou Agricultural Sciences
基金
福建省自然科学基金项目(2020J01181)。
关键词
非洲猪瘟病毒
E165R基因
环介导等温扩增
快速检测
特异性
灵敏性
African swine fever virus(ASFV)
E165R gene
loop-mediated isothermal amplification(LAMP)
rapid detection
specificity
sensitivity
