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

Development and application of real-time fluorescent quantitative RT-PCR method for the detection of Pineapple mealybug wilt associated virus-1

菠萝凋萎相关病毒-1(Pineapple mealybug wilt associated virus-1,PMWaV-1)是田间检出率最高的菠萝凋萎病毒。本研究根据PMWaV-1CP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。

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＆#215;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.