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作 者:廖帆 谭磊[1] 雷磊 湛洋[1] 段德勇[1] 王爱兵[1] LIAO Fan;TAN Lei;LEI Lei;ZHAN Yang;DU AN De-yong;WANG Ai-bing(College of Veterinary Medicine,Hunan Agricultural University,Changsha 41012S,China)
机构地区:[1]湖南农业大学动物医学院,湖南长沙410128
出 处:《中国兽医科学》2022年第6期679-684,共6页Chinese Veterinary Science
基 金:湖南省自然科学基金项目(2020JJ4041);湖南省研究生科研创新项目(CX2020065)。
摘 要:为建立一种适用于猪圆环病毒2型(PCV2)现场、快速、可视化检测方法,本研究根据PCV2ORF1基因中的保守序列设计合适的环介导等温扩增(LAMP)引物对,优化反应体系及条件,并评估该方法的灵敏度和特异性。结果显示,63℃恒温扩增45 min即可出现梯形条带,检测下限为1.0×10^(1)copies/μL,较普通PCR敏感性提高100倍,且当产物中加入SYBR GreenⅠ染料后可实现可视化检测。应用该方法对72份临床样品进行检测,结果显示,PCV2阳性率为47.2%(34/72),与qPCR检测的符合率为98.6%(71/72),与普通PCR的符合率为94.4%(68/72)。结果表明,该基于LAMP的检测方法具有敏感特异、简便快捷、可视化的特点,为PCV2临床检测提供了一种新的选择。In order to establish a field,rapid and visual detection method for porcine circovirus types 2(PCV2),loop-mediated isothermal nucleic acid amplification(LAMP)-based primers were designed according to the conserved sequence of PCV2 ORF1 gene.Subsequently,the reaction system and conditions,the sensitivity and specificity of this detection method were optimized and determined,respectively.The results showed that ladder strips were observed after 45 min at 63℃.The detection limit of this approach reached to 1.0×10^(1)copies/μL,which was 100 times higher than that of the ordinary PCR(1.0×103copies/μL),and the LMAP products stained with SYBR Green I were visualized.A total of 72 suspected clinical samples were tested by this method with a positive rate of 47.2%(34/72),which showed high coincidences with real-time PCR and ordinary PCR,being 98.6%(71/72)and 94.4%(68/72)respectively.The results indicated that this LAMP-based approach are sensitive,specific,simple,rapid,and visual,thereby providing a novel option for PCV2 detection.
关 键 词:猪圆环病毒2型 ORF1基因 环介导等温扩增技术 可视化
分 类 号:S852.659.2[农业科学—基础兽医学]
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