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基于 TaqMan 实时 PCR 和 LAMP 的 FTP-LAMP 检测方法的开发,用于特异性检测植物和昆虫媒介中的韧皮部坏死病菌 De Donno 和桑树菌株。

Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors.

机构信息

Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9, 70010 Valenzano, BA, Italy.

Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9, 70010 Valenzano, BA, Italy.

出版信息

J Microbiol Methods. 2020 Aug;175:105992. doi: 10.1016/j.mimet.2020.105992. Epub 2020 Jun 23.

Abstract

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).

摘要

我们开发了两种实时检测方法,TaqMan 实时 PCR 和 LAMP,使用基于编码 Xylella fastidiosa(Xf)血凝素相关蛋白(Hg)序列设计的引物和探针,该基因仅存在于意大利橄榄(De Donno 橄榄)和美国桑树品系中,用于特异性检测目标 Xf 菌株。这些检测方法使用从 Xf 感染植物样本和两种昆虫载体(Philaenus spumarius,Ps;和 Neophilaenus campestris,Nc)中提取的 DNA 样本进行了验证。这两种技术均被证明具有高度敏感性(100 fg Xf 基因组 DNA),并且仅针对意大利 De Donno 和美国桑树品系的 Xf 具有特异性。当我们的 LAMP 以双重方式与先前发表的用于检测所有 Xf 亚种和菌株的通用引物和探针结合使用时,该双重 LAMP 显示出在同时检测和区分意大利 De Donno 和美国桑树株系与其他亚种/菌株方面的高度多功能性。此外,Hg 基因特异性 LAMP 引物和 TaqMan 探针被用于开发一种新方法;此后称为淬灭后 TaqMan 探针的荧光-环介导等温扩增(FTP-LAMP)。在 FTP-LAMP 中,将 Xf-Hg 特异性荧光猝灭探针添加到标准 LAMP 反应中,仅在与靶标结合时才会发出荧光,从而允许在 LAMP 背景下对 Xf-意大利 De Donno 和美国桑树株系进行序列特异性检测。我们的 FTP-LAMP 检测方法在测试从感染植物、DAS-ELISA 粗汁液和昆虫载体中提取的 Xf 基因组 DNA 时,显示出高度的敏感性,可检测到低至 100 fg 的 Xf 基因组 DNA。此外,当应用于来自昆虫载体的 DNA 模板时,该检测方法具有很高的特异性(98.7%比 LAMP 的 89%)。通过添加一个额外的靶序列特异性探针作为直接的 Xf 特异性染料,FTP-LAMP 具有更高的特异性,并减少了 LAMP 检测法的一个主要问题(假阳性),当用于检测昆虫载体中的 Xf 时。据我们所知,本研究报告了第一种 LAMP 检测方法和第一种新型 FTP-LAMP 方法的开发,用于特异性检测意大利 De Donno 和美国桑树品系的 Xf。与双重方法中的 Xf 通用 LAMP 引物一起,FTP-LAMP 不仅可以成为特异性检测意大利橄榄相关菌株的有用工具,还可以区分已在意大利和欧洲(德国、法国、西班牙和葡萄牙)报道的 Xf 其他菌株(德国、法国、西班牙和葡萄牙)。

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