为了快速准确检测洋葱腐烂病菌(Burkholderia gladioli pv.alliicola,简称Bga),根据GenBank中Bga与相关种的序列差异设计特异引物BG5/BG7和探针Bga-P,建立了 Bga常规PCR和荧光PCR检测方法.测试结果表明,引物和探针对供试的11株Bga菌株表现为阳性反应,其他64株供试菌株和空白对照均为阴性.常规PCR和荧光PCR方法的检测灵敏度分别为24 pg和240 fg菌体DNA.美国、法国、意大利等国进境的80批次洋葱种子样品的检测结果显示,这两种方法的阳性检出率分别为7.5％和12.5％.选取阳性样品进行病菌分离,成功从2批次法国进境洋葱种子中分离到目的菌.本文建立的洋葱腐烂病菌PCR检测方法可为口岸检测部门提供更高效、灵敏和特异的检测手段.

玉米细菌性条斑病是一种对玉米造成严重危害的新型细菌性病害,其病原为Xanthomonas vasi-cola pv.vasculorum,近10年间传播迅速,从非洲蔓延至南北美洲,目前分布于美国、巴西、阿根廷和南非等国家.本文对玉米细菌性条斑病的病原分类地位、危害症状、寄主、地理分布、传播途径、侵染循环、检测方法以及防控措施进行简述,以降低病菌随贸易传入我国的风险.

马铃薯斑纹片病菌(Candidatus Liberibacter solanacearum)是一种主要为害伞形花科和茄科植物的重要有害生物,目前无法人工培养且可随种子远距离传播.准确灵敏的PCR检测方法对病害预警和防止扩散具有重要意义.本文通过复合引物法构建扩增内标(internal amplification control,IAC),建立了马铃薯斑纹片病菌含扩增内标的双重实时荧光PCR检测体系,有效指示了PCR检测过程中的假阴性现象.试验结果表明:双重与单重实时荧光PCR的检测灵敏度一致,当扩增内标添加量为3.4×10-5ng时,样品DNA的检测低限为0.4 ng.40个样品的检测结果显示,与单重实时荧光PCR相比,双重荧光PCR方法的阳性检出率由25.00％提高到27.50％,提高了检测结果的准确性.

为了快速、准确地鉴定番茄斑萎病毒,根据GenBank中韩国的番茄斑萎病毒株系(序列号:KC261961)ssRNA-S基因组编码保守外壳蛋白基因序列设计特异性引物TSWV 5F/5R,建立了番茄斑萎病毒RT-RPA检测方法.特异性检测结果表明,仅番茄斑萎病毒样品扩增出215 bp目标条带,而其他4种病毒样品无扩增出目标条带.灵敏度检测结果表明番茄斑萎病毒RPA检测方法可以达到10-5 cDNA稀释度.

南方菜豆花叶病毒(Southern bean mosaic virus,SBMV)是我国禁止进境的检疫性有害生物.应用RT-PCR和实时荧光RT-PCR方法,从西班牙进境的菜豆种子中检疫鉴定出该病毒,这是我国首次在西班牙进境菜豆种子中截获该病毒.

Thaumatotibia leucotreta is a worldwide important quarantine pests, which has caused a great harm to fruit and crop production, and had impact on international trade. In this study, we amplified the COI fragment from the Tortricidae samples which were intercepted in avocados carried by a tourist from Garner at Guangzhou Baiyun Airport in January of 2014. We sequenced the fragments and blasted in the GenBank, Barcoding of Life Data Systems (BOLD). A neighbor-joining tree was constructed. The intercepted insects were identified as Thaumatotibia leucotreta based on the DNA barcodes results. Our results demonstrate that, the DNA barcode could be used for quick and accurate identification of Thaumatotibia leucotreta.

The fast identification methods are developed to shorten the time for identification of the quarantined fruit flies.This review describes the development of the fast identification methods,including electrophoresis,restriction fragment length polymorphism (RFLP),random amplified polymorphic DNA (RAPD) and nucleic acid sequences analysis for the quarantined fruit fly.These methods were compared and the suitable molecular markers are offered to provide basic ideas for rapid identification of the quarantined fruit flies.