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

作　　者：胡加谊[1,2] 罗志文[1] 李向宏[1] 范鸿雁[1] 周朋[3] 章绍延[3] 张治礼[1] 刘志昕[3] 何凡[1] 

机构地区：[1]海南省农业科学院热带果树研究所/农业部海口热带果树科学观测实验站,海口571100 [2]海南省农垦科学院,海口570206 [3]中国热带农业科学院热带生物技术研究所农业部热带作物生物学与遗传资源利用重点实验室,海口571101

出　　处：《植物保护》2014年第6期116-121,125,共7页Plant Protection

基　　金：公益性行业(农业)科研专项(201203021);海南省重大科技项目(ZDZX2013020-3);海南省科学事业费项目(琼财预[2013]131号);海南省重点科技计划应用研究及产业化项目(ZDXM20130031);海南省自然科学基金(琼科[2013]32号)

摘　　要：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.

关 键 词：菠萝凋萎相关病毒-1 TAQMAN探针 实时荧光定量RT-PCR 检测 

分 类 号：S436.67[农业科学—农业昆虫与害虫防治]