多重PCR快速检测槟榔黄化植原体和槟榔隐症病毒1体系的建立

Establishment of a multiplex PCR system for the rapid detection of areca palm yellow leaf phytoplasma and areca palm velarivirus1

由槟榔黄化植原体(areca palm yellow leaf phytoplasma,AYLP)引起的黄化病和槟榔隐症病毒1(Areca palm velarivirus 1,APV1)引起的黄叶病毒病是危害槟榔树最严重的两种病害,对海南槟榔产业造成了巨大的经济损失,建立快速的检测技术对于两种病害的预警防控具有重要意义。利用多重PCR(Multiplex PCR)技术同时检测AYLP和APV1,即AYLP巢式PCR第一轮结束后,再使用一次多重PCR对两种病原同时进行扩增,最后进行电泳观察结果。结果显示,通过对多重PCR浓度体系和退火温度进行优化,在一个体系中成功扩增出AYLP和APV1,得到525和311 bp两条特异性大小条带。多重PCR检测与单一PCR检测结果完全一致,并且与单一PCR检测方法比较,多重PCR方法精简了操作步骤,提高了检测效率,显著降低了检测成本,为槟榔黄化病和黄叶病毒病的常规诊断提供了新方法。因此,该技术在AYLP和APV1常规检测中具有重要的应用价值。

Yellow leaf disease caused by areca palm yellow leaf phytoplasma(AYLP)and the yellow leaf vi-rus disease caused by Areca palm velarivirus 1(APV1)are two of the most serious diseases affecting areca palm trees,causing huge economic losses to the areca palm industry in Hainan,and the establishment of rapid detection techniques is of great importance for the early warning and prevention and control of both diseases.In this study,multiplex PCR was used to detect areca AYLP and APV1 simultaneously,i.e.after the first round of nested PCR for AYLP,the multiplex PCR was used to amplify the two pathogens simultaneously,and the results were finally observed by electrophoresis.As results,by optimizing the multiplex PCR concentration system and annealing temperature,AYLP and APV1 were successfully amplified in one system,and two specific size bands of 525 bp and 311 bp were obtained.The results of multiplex PCR were in complete agreement with the results of single PCR,and compared with the single PCR assay,the multiplex PCR method streamlined the operation steps,im-proved the detection efficiency and significantly reduced the detection cost,providing a new method for the rou-tine diagnosis of areca palm yellow leaf disease and yellow leaf virus diseases.Therefore,this technique has im-portant applications in the routine detection of AYLP and APV1.