检测鹅细小病毒环介导等温扩增方法的建立及结果判定方法改进

Development of Loop-mediated Isothermal Amplification Method and Improvement of Result Determination Method for the Detection of Goose Parvovirus

摘要: 利用新兴的环介导等温扩增技术,建立了一种灵敏、特异、快速的体外环介导等温扩增检测方法。针对鹅细小病毒VP3基因的8个位点设计了6条特异性引物,建立了一种快速检测鹅细小病毒的LAMP方法。试验结果表明LAMP方法在恒温65℃水浴下50min内即可完成GPV的检测,且其检测灵敏度达150pg.μL-1;与其他常见的病毒无交叉反应。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,可见该方法为适合基层及现场快速检测、实用灵敏的分子生物学方法。

Abstract: A novel speed LAMP assay for GPV was explored with Loop-mediated amplification(LAMP).With two pairs of identify six positions in VP3 gene,a LAMP assay was developed for the rapid detection GPV.The results indicated that GPV was detected by LAMP assay at 65℃ as early as 50 min of incubation in a simple water bath.The detection limit of the LAMP assay was 150 pg·μL-1 of GPV genome.And no amplification was found with the samples of other viruses.A mixture of calcein and Mn2+ was used as fluorescent reagent to make the result of LAMP visible.Therefore,the simple and fast method is a potential useful technique for GPV detection in the field.