大麦条纹花叶病毒荧光定量PCR检测方法的建立及应用

Establishment and application of fluorescence quantitative PCR detection method for Barley stripe mosaic virus

大麦条纹花叶病毒Barley stripe mosaic virus(BSMV)是我国进境植物检疫性有害生物。为提高大麦条纹花叶病毒检测的灵敏度和特异性,缩短检测周期,根据该病毒不同分离株外壳蛋白(coat protein,CP)基因的保守序列设计特异性的引物和探针,建立了BSMV的实时荧光RT-PCR检测方法。结果表明,本检测方法特异性强,对南方菜豆花叶病毒Southern bean mosaic virus(SBMV)、小麦线条花叶病毒Wheat streak mosaic virus(WSMV)、玉米褪绿斑驳病毒Maize chlorotic mottle virus(MCMV)、烟草环斑病毒Tobacco ringspot virus(TRSV)、菜豆荚斑驳病毒Bean pod mottle virus(BPMV)和番茄环斑病毒Tomato ringspot virus(ToRSV)6种病毒均无交叉扩增;且检测方法灵敏度高,对RNA模板的最低检测限达到5×10^(-4) ng/μL,与双抗体夹心酶联免疫吸附法(DAS-ELISA)和普通RT-PCR相比,本方法的灵敏度分别提高了10倍和100倍。此外,我们利用该方法对12批进口大麦进行检测,发现6批大麦中检出大麦条纹花叶病毒,占比约50%。总之,本研究建立的荧光定量PCR方法具有灵敏度高、特异性强等优点,适合于BSMV的快速检测。

Barley stripe mosaic virus(BSMV)is a quarantine pest in China.In order to improve the detection sensitivity,specificity and shorten the detection cycle,based on the conserved sequences in coat protein genes of different BSMV isolates,we established a real-time fluorescent RT-PCR detection method with specific primers and TaqMan probe.The results showed that this method could be only used for BSMV,but not Southern bean mosaic virus(SBMV),Wheat streak mosaic virus(WSMV),Maize chlorotic mottle virus(MCMV),Tobacco ring spot virus(TRSV),Bean pod mottle virus(BPMV)or Tomato ringspot virus(ToRSV).The minimum detection limit of template RNA was 5×10^(-4) ng/μL.Compared with double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)or ordinary RT-PCR,the sensitivity was increased by 10 or 100 times,respectively.In addition,BSMV was detected in six of 12 batches of imported barley with this method.The real-time PCR method is a rapid,sensitive and highly specific method for BSMV.