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Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen Erwinia amylovora

机译:常规和实时荧光定量PCR检测Erwinia piriflorinigrans使其与火疫病病原体Amylova amylovora有所区别

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摘要

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 103 cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 102 cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.
机译:Erwinia piriflorinigrans是欧文氏菌属的一种新病原体,最近已在西班牙进行了描述。准确检测和鉴定E. piriflorinigrans具有挑战性,因为它在梨花上的症状与火疫病的致病因子Amwinlov amylovora引起的症状相似。而且,这两个物种具有表型和分子特征。开发了两种特异性和灵敏的常规PCR和实时PCR方案,以鉴定和检测piriflorinigrans,并将其与支链淀粉和该属的其他物种区分开。这些协议基于质粒pEPIR37的序列,该序列存在于所分析的所有E. piriflorinigrans菌株中。在证明了质粒的稳定性之后,通过扩增所有测试的E. piriflorinigrans菌株证实了该方案的特异性,而未扩增来自梨树的304个密切相关的致病性和非致病性欧文氏菌菌株和微生物群。在敏感性分析中,通过常规或实时PCR在加标植物材料中检测到10 3 细胞/ ml提取物,在从植物中提取的DNA中检测到10 2 细胞/ ml通过实时PCR检测加标的植物材料。此处开发的协议成功地从坏死的火棘属植物中的564个有症状和无症状的自然感染梨样本(花朵,皮层茎组织,叶片,枝条和小果)中的102个中成功检测到了E. piriflorinigrans。开花,以及在坏死的梨和苹果组织中都感染了支链球菌和piriflorinigrans。因此,这些新工具可用于流行病学研究,这将加深我们对不同宿主和植物组织中埃里希菌的生命周期及其与支链淀粉菌相互作用的了解。

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