基于重组酶聚合酶扩增技术(RPA)的柑橘溃疡病菌检测方法  

The Rapid Detection of Xanthomonas citri ssp.citri(Xcc)Based on Recombinase Polymerase Amplification(RPA)Assay

作　　者：马志敏[1] 段玉 许建建 宾羽 周常勇[1] 宋震[1] MA Zhimin;DUAN Yu;XU Jianjian;BIN Yu;ZHOU Changyong;SONG Zhen(Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,Chongqing 400712,China)

机构地区：[1]西南大学,中国农业科学院柑桔研究所,重庆400712

出　　处：《园艺学报》2021年第3期590-599,共10页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2017YFD0202002);国家现代农业产业技术体系建设专项资金项目(CARS2605B)。

摘　　要：根据柑橘溃疡病菌(Xanthomonas citri ssp.citri,Xcc)基因组的保守序列设计特异引物,通过对引物浓度、反应温度和反应时间条件优化,建立了柑橘溃疡病菌重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)检测方法。该方法无需PCR仪等复杂设备,在39℃恒温反应30 min即可完成检测过程,快速简便。该检测方法与其他柑橘病原无交叉反应,特异性强;检测灵敏度是普通PCR的100倍,与实时荧光定量PCR基本一致。对71个柑橘样品进行检测,RPA检测出溃疡病阳性样品22个,与PCR、实时荧光定量PCR检测结果一致。A detection assay based on recombinase polymerase amplification(RPA)for Xanthomonas citri ssp.citri(Xcc)was established by designing specific primers and optimizing concentration of primers,reaction temperature and reaction time.The method requires no complex equipment such as PCR apparatus,and can complete the detection process at 39℃in 30 min,which is quick and simple.The detection method has strong specificity to Xcc with no cross reaction with other citrus pathogens.The RPA detection sensitivity was 100 times higher than that of ordinary PCR,which was the same as that of real-time quantitative PCR.In 71 citrus samples,22 samples were positive to canker detected by RPA,which was consistent with the results of PCR and real-time quantitative PCR.

关 键 词：柑橘溃疡病 重组酶聚合酶扩增 RPA 检测 

分 类 号：S666[农业科学—果树学]