基于PfAgo技术的猪细小病毒快速检测方法的建立  

Establishment of a rapid detection method for porcine pavovirus based on PfAgo technology

作　　者：吴崇铤 苟婧萱 兰守信 张曼琪 涂健[1] 王振宇 邵颖[1] 宋祥军 WU Chongting;GOU Jingxuan;LAN Shouxin;ZHANG Manqi;TU Jian;WANG Zhenyu;SHAO Ying;SONG Xiangjun(Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control,School of Veterinary Medicine,Anhui Agricultural University,Hefei 230036)

机构地区：[1]安徽农业大学动物医学院/兽医病理生物学与疫病防控安徽省重点实验室,合肥230036

出　　处：《安徽农业大学学报》2024年第6期1013-1021,共9页Journal of Anhui Agricultural University

基　　金：2023年国家级大学生创新创业训练计划项目(X202210364309,X202210364411);2023年安徽农业大学校级大学生创新创业训练计划项目(X202310364040)共同资助。

摘　　要：为了将优化的PfAgo荧光法与环介导等温扩散(loop-mediated isothermal amplification,LAMP)技术相结合,建立灵敏、快速、特异的猪细小病毒(porcine pavovirus,PPV)检测方法,针对PPV保守序列VP2分别筛选gDNA并设计对应的分子信标,初步建立基于PfAgo的荧光检测法,在同靶位筛选LAMP的引物并对其扩增条件进行优化,将两者结合探究其敏感性和特异性,最后使用优化后的方法对临床样本进行检测。结果表明PfAgo荧光检测法最佳反应温度为95℃,最佳引物使用量为750 pmol,分子信标最佳使用量为15 pmol,最佳反应时间为30 min,PfAgo荧光检测法具有高度的专一性。LAMP检测法筛选出最佳扩增温度为61℃,最适反应时间为40 min,dNTP最佳终浓度为1.2 mmol·L^(-1),锰离子最佳终浓度为10 mmol·L^(-1),最适内外引物比例为4∶1,其灵敏度可达101 copies·µL^(-1),与其他检测样本无交叉反应、特异性良好。将LAMP和PfAgo荧光检测法相结合后,其灵敏度可达101 copies·µL^(-1),特异性试验结果表明该方法专一性强。研究建立的PPV-LAMP-PfAgo荧光法,灵敏性高、特异性强,具备灵敏、快速等优点,为临床基层检测PPV提供一种新的技术手段。In order to establish a sensitive,rapid,and specific detection method for porcine parvovirus(PPV)by combining the optimized PfAgo fluorescence method with loop-mediated isothermal amplification(LAMP)technology,gDNA was screened for the conserved sequence of the VP2 gene of PPV,and corresponding molecular beacons were designed to preliminary establish a PfAgo based fluorescence detection method.Primers for LAMP were screened at the same target locus and their amplification conditions were optimized.The combination of the two methods were then investigated for sensitivity and specificity,and the optimized method was ultimately used for the detection of clinical samples.The results showed that the optimal reaction temperature for the PfAgo fluorescence assay was 95°C,with an optimal primer dosage of 750 pmol,an optimal molecular beacon dosage of 15 pmol,and an optimal reaction time of 30 minutes.The PfAgo fluorescence assay demonstrated high specificity.The LAMP assay identified an optimal amplification temperature of 61°C,an optimal reaction time of 40 minutes,an optimal final concentration of dNTP of 1.2 mmol·L^(-1),an optimal final concentration of manganese ion of 10 mmol·L^(-1),and an optimal ratio of internal to external primers of 4∶1.Its sensitivity was as low as 101 copies·μL^(-1),with no cross-reaction with other samples,indicating good specificity.When LAMP was combined with PfAgo fluorescence detection method,the sensitivity achieved at 101 copies·μL^(-1),and specificity assay results indicated that the method was highly specific.The PPV-LAMP-PfAgo fluorometric method established in this study offers high sensitivity and specificity,with the advantages of sensitivity and rapidity.It provides a new technical approach for the detection of PPV at the clinical grassroots level.

关 键 词：PfAgo 猪细小病毒 环介导等温扩增 快速检测 

分 类 号：S858.28[农业科学—临床兽医学]