黄瓜花叶病毒荧光定量 PCR 检测方法的建立

孙 洁; 王 婉; 周 翎; 阮小蕾; 饶雪琴; 李华平

doi:10.7671/j.issn.1001-411X.2014.02.010

黄瓜花叶病毒荧光定量 PCR 检测方法的建立

Real-time PCR for quantification of Cucumber mosaic virus in banana

摘要

摘要: 【目的】建立检测香蕉中黄瓜花叶病毒 Cucumber mosaic virus（CMV）的实时荧光定量方法.【方法】根据 CMV 外壳蛋白（CP）保守序列设计了 TaqMan 实时荧光定量 PCR 特异性探针及引物，优化反应体系检测 TaqMan 探针实时荧光定量方法的灵敏度、特异性和重复性.【结果和结论】该方法检测灵敏度为4.2×10² μL^-1，比普通PCR高100倍，且与香蕉束顶病毒 Banana bunchy top virus（BBTV）、香蕉线条病毒 Banana streak virus（BSV）无交叉反应，特异性和重复性都较好.用实时荧光定量 PCR 检测 14 份田间香蕉样品有 5 份样品为阳性，进一步证明建立的实时荧光定量方法可用于香蕉 CMV 的检测.

Abstract: 【Objective】To develop a real-time quantitative PCR method with TaqMan probes to quantify Cucumber mosaic virus(CMV) in banana.【Method】The primers and probes were designed based on the conserved coat protein(CP) sequences of CMV and were applied to real-time PCR assays.The reaction system was optimized,and its sensitivity, specificity and repeatability were evaluated.【Result and conclusion】The detection sensitivity of the real-time PCR assay was 4.2×10² μL^-1, which was 100 times more sensitive than PCR. The specifity of the assay was analyzed with Banana bunchy top virus(BBTV) and Banana streak virus(BSV), and no cross reaction were observed. The assay also had good repetitions. The real-time PCR assay was evaluated with field samples. 5 of the 14 tissue samples collected from field suspected CMV infected bananas were positive, which further confirmed that the real-time PCR method should be suitable for detection and quantitation of CMV in banana.

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