菠萝凋萎相关病毒-1实时荧光定量RT-PCR检测体系的建立与应用

摘要

Pineapple mealybug wilt associated virus-1 (PMWaV-1 )highly infected pineapple in growing region in Hainan Province. A method of real-time fluorescent quantitative RT-PCR was established and optimized by using TaqMan probe and specific primers according to conserved sequence from the coat protein(CP)gene of PMWaV-1 . The standard curve of the method was y= -3.307×log x+38.18,r2= 0.998. The results showed that the meth-od was specific to PMWaV-1 and minimum detectable copies were 40. Both the variation coefficient of intra-assay and inter-assay for the method were less than 1.98% . Therefore,the established real-time fluorescent quantitative RT-PCR is a sample,specific,sensitive and reproducible method for PMWaV-1 detection. Detection analysis indi-cated that the content of PMWaV-1 in old leaves,younger leaves and suckers went down gradually.%菠萝凋萎相关病毒-1（Pineapplemealybugwiltassociatedvirus-1，PMWaV-1）是田间检出率最高的菠萝凋萎病毒。本研究根据PMWaV-1 CP基因保守序列设计特异性TaqMan探针和引物，建立并优化了PMWaV-1实时荧光定量RT-PCR检测方法。优化后的反应体系制备的标准曲线为y＝－3．307×log x＋38．18，相关系数r2为0．998。试验结果表明，该方法能特异性地检测PMWaV-1，对PMWaV-2、3和阴性对照均无反应；最低检测限达到40拷贝。重复性试验表明批内和批间变异系数均小于1．98％，是一种操作简便、特异性强、灵敏度高、重复性较好的PMWaV-1定量检测方法。样品检测结果表明PMWaV-1在菠萝植株老叶、嫩叶和吸芽中的病毒含量呈递减趋势。