We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).

我们使用引物和探针开发了两种实时检测测定法，即TaqMan实时PCR和LAMP，该引物和探针的设计基于一个序列，该序列被标注为编码Xylella fastidiosa（Xf）的血凝素相关蛋白（Hg）的一种基因，该基因专门存在于意大利橄榄（De Donno of Olive）和美国桑树菌株，用于目标Xf菌株的特异性检测。用从Xf感染的植物样品和两种昆虫载体（Philaenus spumarius，Ps ;和Neophilaenus campestris，Nc）提取的DNA样品验证了这些测定的有效性。两种技术均被证明具有很高的灵敏度（100 fgXf基因组DNA），并且特指意大利De Donno和美国桑树Xf菌株。当将我们的LAMP与先前发布的通用引物和探针结合用于双链检测所有Xf亚种和菌株时，双链LAMP在同时检测和区分意大利De Donno和美国桑树染色形式时表现出很高的通用性其他亚种/菌株。此外，利用Hg基因特异性LAMP引物和TaqMan探针开发了一种新方法。此后简称˚F的luorescence Ť aqMan P在去淬灭长袍-大号接力介导的等温放大器简化（FTP-LAMP）。在FTP-LAMP中，将Xf - Hg特定的荧光团猝灭探针添加到标准LAMP反应中，并且仅在与靶标结合时发出荧光，从而可以对Xf -Italian De Donno和美国桑树菌株进行序列特异性检测。LAMP上下文。我们的FTP-LAMP法显示为检测到100 FG的基因组DNA高度敏感Xf中，当上测试Xf中从感染的植物，DAS-ELISA的粗液汁及昆虫媒介萃取-genomic DNA。此外，当将其应用于昆虫载体的DNA模板时，该测定法显示出高特异性（LAMP为98.7％，而LAMP为89％）。通过添加额外的靶序列特异性探针作为直接Xf作为一种特定的染料，当用于检测昆虫载体中的Xf时，FTP-LAMP具有更高的特异性并减少了LAMP分析的主要问题之一（假阳性）。据我们所知，本研究报告了第一个LAMP测定法和第一个新颖的FTP-LAMP方法的开发，该方法可用于特异性检测意大利De Donno和美国桑树Xf。FTP-LAMP与Xf通用LAMP引物以双工方式一起使用，不仅可以作为有用的工具，不仅可以用于意大利义大利橄榄相关菌株的特异性检测，还可以将De Donno菌株与已经存在的其他Xf菌株区分开来在意大利和欧洲（德国，法国，西班牙和葡萄牙）进行了报道。