柑橘溃疡病qPCR检测方法的验证

Q3 Agricultural and Biological Sciences

New Zealand Plant Protection Pub Date : 2019-07-28 DOI:10.30843/NZPP.2019.72.328

H. Lee, W. Ho, R. Thangavel, J. Baskarathevan, B. Alexander

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引用次数: 1

摘要

柑橘溃疡病是由柑橘黄单胞菌引起的一种严重影响柑橘产业的细菌性疾病。柑橘(Xcc)致病型A, A*和Aw，并在较小程度上由X. fuscans亚sp。aurantifolii (Xfa)。最近在澳大利亚爆发的柑橘溃疡病强调需要重新评估用于检测柑橘溃疡病细菌的分子分析方法的效率。两种已发表的针对pth和lrp基因的基于探针的qPCR检测Xcc，而基于SYBR green的qPCR检测Xfa。Xcc pth基因和Xfa qPCR分别对Xcc和Xfa的所有病型具有特异性。两种检测方法的检出限均为1 pg基因组DNA或103 CFU细菌叶片样品。Xcc lrp基因qPCR检测方法能够区分Xcc病原菌，检出限为1 ng基因组DNA或106 CFU，但该方法与Xfa存在交叉反应。为了快速、高通量地检测所有Xcc亚型，我们将COX引物作为pth基因qPCR检测的内控物，建立了一种基于双探针的qPCR检测方法。

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Validation of qPCR assays for the detection of citrus canker

Citrus canker, a serious bacterial disease affecting the citrus industry worldwide, is caused by Xanthomonas citri subsp. citri (Xcc) pathotypes A, A* and Aw, and to a lesser extent by X. fuscans subsp. aurantifolii (Xfa). The recent citrus canker outbreak in Australia has emphasised the need to re-evaluate the efficiency of molecular assays used for detecting citrus canker bacteria. Two published probe-based qPCR assays targeting the pth and lrp genes were tested for Xcc, whereas a SYBR Greenbased qPCR assay was tested for Xfa. The Xcc pth gene and Xfa qPCR assays were shown to be specific towards all pathotypes of Xcc and Xfa, respectively. The detection limit for both assays were 1 pg of genomic DNA or 103 CFU in bacteria-spiked leaf sample. The Xcc lrp gene qPCR assay was able to discriminate Xcc pathotypes with a detection limit of 1 ng of genomic DNA or 106 CFU in bacteriaspiked leaf sample, but this assay showed cross-reaction with Xfa. To allow rapid high-throughput detection of all Xcc pathotypes, a duplex probe-based qPCR assay was developed by incorporating COX primers as an internal control for plant DNA into the pth gene qPCR assay.

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来源期刊

New Zealand Plant Protection Agricultural and Biological Sciences-Insect Science

CiteScore

1.10

自引率

0.00%

发文量

期刊介绍： New Zealand Plant Protection is the journal of the New Zealand Plant Protection Society. It publishes original research papers on all aspects of biology, ecology and control of weeds, vertebrate and invertebrate pests, and pathogens and beneficial micro-organisms in agriculture, horticulture, forestry and natural ecosystems of relevance to New Zealand.