菠萝泛菌与水稻白叶枯病菌双重PCR检测方法的建立与应用

Development and application of a duplex PCR assay for the detection of Xanthomonas oryzae pv.oryzae and Pantoea ananatis

近年来水稻发生了一种由菠萝泛菌Pantoea ananatis引起的新型细菌病害,其发生时期与白叶枯病相近,病症与白叶枯病类似。为了实现该病害与白叶枯病的快速检测,本研究基于泛菌属看家基因acnA,通过序列比对,设计了22对特异性引物,分别与已知的白叶枯病菌检测引物XOO80进行配对并筛选,建立了一种双重PCR检测方法,可从菠萝泛菌和白叶枯病菌中分别扩增出910 bp和162 bp的特异性条带,而其他10种非目标细菌均未有扩增条带,25μL体系中可稳定地从至少1 pg/μL的基因组DNA模板中扩增出特异性条带。本研究建立的菠萝泛菌与水稻白叶枯病菌双重PCR检测方法具有良好的特异性和灵敏度,为水稻细菌病害病原鉴定及防治提供了技术支撑。

In recent years,a new bacterial disease caused by Pantoea ananatis occurred in rice.Occurrence period and symptom of this disease were similar to those of rice bacterial leaf blight.To rapidly detect and distinguish the causing agent of the new bacterial disease and rice bacterial leaf blight,22 pairs of specific primers were designed based on the housekeeping-gene acnA in Pantoea through sequence alignment.Specific primers were paired with primer set XOO80,which was used for detection of Xanthomonas oryzae pv.oryzae.After screening,a duplex PCR assay was developed,and bands with the size of 910 bp and 162 bp was specifically amplified from P.ananatis and X.oryzae pv.oryzae,respectively,whereas no band was amplified from the other 10 non-target strains.In a 25μL system,specific bands could be stably amplified from genomic templates at least 1 pg/μL.The duplex PCR assay for the simultaneous detection of P.ananatis and X.oryzae pv.oryzae in this study was specific and sensitive,which will provide reliable technical support for identification of pathogen and control of rice bacterial diseases.