猪细小病毒LAMP-LFD检测方法的建立及初步应用

Establishment and preliminary application of LAMP-LFD method for detection of Porcine parvovirus

为了建立一种用于猪细小病毒(Porcine parvovirus,PPV)的特异、灵敏、快速检测方法,试验根据GenBank发布的PPV基因序列保守区片段设计了一套带生物素标签的特异性引物和FITC探针,以pET28a-NS1重组质粒为阳性模板将环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术与侧向流动层析试纸(lateral flow dipstick,LFD)相结合,建立了PPV-LAMP-LFD检测方法,对LAMP-LFD的反应温度、反应时间及胶体金标记FITC-mAb比例进行优化,分析该方法的敏感性和特异性,并检测其早期临床应用效果。结果表明:建立的PPV-LAMP-LFD检测方法最适反应温度为64℃,最佳反应时间为50 min;标记1 mL胶体金需要14μg FITC-mAb;该检测方法的最低检测限为1×10 copies/μL,比琼脂糖凝胶电泳和荧光定量PCR灵敏度高19倍;对猪伪狂犬病毒(PRV)、蓝耳病病毒(PRRSV)、猪圆环病毒2型(PCV-2)、猪乙型脑炎病毒(JEV)和猪瘟病毒(CSFV)检测结果均呈阴性;早期临床应用显示可在猪感染PPV后4 h的血液中检出病毒。说明建立的PPV-LAMP-LFD检测方法具有灵敏、特异、快速、简便等优点。

The purpose of this study was to establish a specific, sensitive and rapid detection method for Porcine parvovirus(PPV). The experiment designed a set of specific primers and FITC probes with biotin tags according to the conserved fragment of PPV gene sequence published by GenBank. A PPV-LAMP-LFD detection method was established using pET28 a-NS1 recombinant plasmid as positive template, loop-mediated isothermal amplification(LAMP) and lateral flow dipstick(LFD) were combined. The reaction temperature and reaction time of LAMP-LFD and the proportion of colloidal gold labeled FITC-mAb were optimized to analyze the sensitivity and specificity of this method, and to detect the effect of its early clinical application. The results showed that the optimal reaction temperature and reaction time of the established PPV-LAMP-LFD detection method were 64 ℃ and 50 min, respectively. Labeling 1 mL colloidal gold required FITC-Amb of 14 μg. The minimum detection limit of the assay was 1×10 copies/μL, and the sensitivity of the assay was nine times higher than that of agarose gel electrophoresis and quantitative PCR. The results for PRV, PRRSV, PCV-2, JEV and CSFV were negative. The virus could be detected in the blood of pigs within 4 h of infection. The PPV-LAMP-LFD detection method was sensitive, specific, rapid and simple.