菠萝凋萎相关病毒–2实时荧光定量RT-PCR检测方法的建立

Development of a Real-time Fluorescent Quantitative RT-PCR Method for the Detection of Pineapple mealybug wilt associated virus-2

菠萝凋萎相关病毒(Pineapple mealybug wilt associated virus-2,PMWaV-2)是引起的菠萝凋萎病(Mealybug wilt of pineapple,MWP)的重要病原。本研究中根据PMWaV-2外壳蛋白基因序列设计特异性引物和探针,建立基于TaqMan探针的实时荧光定量RT-PCR检测方法。该方法能高灵敏检测出阳性样品,对阴性样品及空白对照均无荧光反应;灵敏度比普通PCR高100倍;重复性试验表明批内和批间变异系数均在1.85%以内,表明本方法是一种操作简便、特异性强、灵敏度高、重复性较好的PMWaV-2定量检测方法。

Pineapple mealybug wilt associated virus-2(PMWaV-2)is an important pathogen that causes Mealybug wilt of pineapple(MWP). This study established a real-time quantitative RT-PCR method with TaqMan probe based on specific primers of conserved nucleotide sequence of PMWaV-2 coat protein gene. The results showed that the method is highly sensitive to positive sample,but has no fluorescence signal to health sample and water control. The sensitivity of real-time quantitative RT-PCR is about 100 times higher than regular PCR. Three-time repeats revealed that the coefficients of variation between the intra- and inter-assay were both within 1.85%,indicating a simple,specificity,high sensitivity,and reliable reproducibility detection method to PMWaV-2.