香蕉束顶病毒实时荧光PCR检测方法的建立

Development of TaqMan Real-time PCR Assay of Banana bunchy top virus

根据香蕉束顶病毒(Banana bunchy top virus,BBTV)复制酶基因保守序列设计特异性探针及引物,建立了BBTV实时荧光PCR(Taq Man real-time PCR)检测方法。结果显示,所建立的检测方法在检测质粒DNA标准品和发病香蕉植株总DNA时,其灵敏度均比普通PCR高,且与黄瓜花叶病毒(Cucumber mosaic virus,CMV)和香蕉线条病毒(Banana streak virus,BSV)无交叉反应。同浓度样品进行7次重复性试验,各重复扩增结果所得Ct值的变异系数为0.93%,表明建立的荧光定量PCR检测方法重复性好,Ct值保持稳定。利用所建立的方法检测带毒香蕉吸芽繁殖的组培苗中的BBTV,发现BBTV含量随着继代数的增加而增加,并且组培苗顶部叶片中的含量高于其他部位的叶片。

A TaqMan real-time PCR assay was developed by designing a set of primers and probe based on the conserved replicase gene of Banana bunchy top virus（BBTV）. The results showed that the assay was more sensitive than Endpoint PCR in detecting both the standard DNA plasmid and total DNA extracted from field samples，respectively，and had no cross reaction with two other common viruses，Cucumber mosaic virus（CMV）and Banana streak virus（BSV）in the banana plants. Seven times of reproducibility tests were analyzed and the results showed that Ct coefficient of variation was 0.93% and the Ct value kept stable in each reaction. This TaqMan real-time PCR assay was utilized to detect BBTV concentrations in diseased micropropagated banana shoots，and the results indicated that the concentrations of BBTV in the micropropagated banana shoots of‘Yueyoukang 1’increased along with tissue-cultured generations，which was higher in the top leaves than those in other leaves.