环介导等温扩增技术快速检测猪细小病毒的试验

Detection of porcine parvovirus by loop-mediated isothermal amplification

利用环介导等温核酸扩增技术(LAMP),建立了一种灵敏、特异、快速的猪细小病毒(PPV)检测方法。该方法针对猪细小病毒非结构蛋白(NS-1)基因保守区域设计6条特异引物,在63℃的等温条件下45min即可完成反应。最低检测限量为10拷贝的PPV目的基因,比常规聚合酶链式反应(PCR)方法敏感100倍,并具有良好的特异性。以钙黄绿素和Mn2+作为荧光指示剂,可快速、直观判定反应结果。通过对149份临床样品进行检测比较,LAMP与PCR检出阳性样本数分别为33份和27份,表明LAMP方法阳性检出率高于PCR。

Using the loop-mediated isothermal amplification （LAMP）, a sensitive and specllm method for detection of porcine parvovirus was established. With six specific primers targeting the conserved region of nonstructural protein （NS-1） gene, the LAMP-based assay could be completed within 45 min at 63℃. Detection limit of the assay was found to be 10 copies per reaction determined by using a recombined plasmid containing the target sequence,and that 100-fold higher than that of the PCR assay. In addition, the LAMP was highly specific to porcine parvovirus. A mixture of calcein and Mn2^- was used as fluorescent reagent to make the result of LAMP visible. Furthermore, LAMP and conventional PCR methods were assayed for 149 clinical samples which were suspected to be porcine parvovirus infection. As a result, 22.1% （33/149） and 18.1% （27/149） of the samples were positive on LAMP and PCR analyzes, respectively. The results demonstrated that LAMP was more sensitive than PCR for clinical diagnosis.