柑桔溃疡病菌滚环扩增检测体系的建立

Detection system for Xanthomonas axonopodis pv. citri using Rolling Circle Amplification

根据柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)独有的蛋白基因序列和锁式探针公共连接序列分别设计特异性的锁式探针及其扩增引物,优化系列反应条件,建立了特异性的柑桔溃疡病菌滚环扩增体系。初步检测结果表明该体系能够特异性地检出Xac的菌体细胞及其DNA,而检测不出供试的其它植物病原细菌和柑桔叶面常见的多种附生细菌;对Xac靶片段克隆质粒DNA的检测灵敏度为102copy/μL,对Xac菌悬液的检测灵敏度为20cfu/μL,比常规PCR的检测灵敏度稍高。用滚环扩增技术和常规PCR技术对田间采集的实际样品进行了检测,两种方法的检测结果没有显著差异(P>0.01)。由于锁式探针的公共连接序列对扩增的条件要求一致,本体系的建立可以为植物病原微生物多靶标检测和病害检疫检验提供新的技术支撑。

Padlock probe was designed based on the sequence of the unique hypothetic protein gene in complete genome of Xanthomonas axonopodis pv. cirri （Xac）, and amplification primers ware designed according to the universal linking sequence of padlock probe. Detection system of rolling circle amplification （RCA） was established and optimized. Results show that the system could detect Xac and its DNA specifically, while other plant pathogens and bacteria attached on the surface of citrus leaves could not be detected. This indicates that the detection system had its specificity. The detection sensitivity of RCA was 20 cfu/μL for Xac cells and 10^2copy/μL for cloned DNA fragment, which was slightly higher than the sensitivity of conventional PCR. Leaf samples collected from orange orchards were detected with both RCA and conventional PCR. The result shows that the Xac positive percentage had no remarkable difference between the two methods （P〉0.01）. Because the universal linking sequence in padlock probe can use same amplification condition, the new technology and detection system can be used to detect diverse plant pathogens simultaneously in plant quarantine and disease pre-symptom diagnosis.