Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)
Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)
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DOI：
10.1007/s10658-009-9439-x
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发表时间：
2009-08-01
影响因子：
1.8
通讯作者：
Wang, N.
中科院分区：
文献类型：
--
作者：
Trivedi, P.;Sagaram, U. S.;Wang, N.
柑橘黄龙病是柑橘的一种毁灭性病害，已知与一种苛养的、韧皮部限制的革兰氏阴性细菌有关，但尚未被培养的黄龙杆菌属细菌。在本研究中，我们已经建立了一种方法来定量活的黄龙病黄龙杆菌亚洲（拉斯维加斯）的援助，乙锭单叠氮（EMA），可以区分活细胞和死细胞。首先，借助于定量实时PCR（QPCR），通过使用由rplKAJL-rpoBC（β-操纵子）区域的703 bp DNA片段组成的质粒模板，形成校准曲线。然后开发标准方程来量化柑橘、长春花和亚洲柑橘木虱Diaphorina citri中的Las基因组当量。为了克服定量PCR在区分细菌活细胞和死细胞方面的局限性，使用EMA抑制植物样品中Las死细胞的DNA扩增。通过使用标准方程和EMA-QPCR方法，我们发现柑橘和长春花中的活细胞比例分别为17-31%和16- 28%。通过定量有症状和无症状叶片中的Las群体，确定HLB症状发展所需的最小细菌浓度。在本研究中开发的EMA-QPCR方法应该提供一个准确的评估可行的HLB病原体，提供了一个工具，调查疾病流行病学，从而作为一个重要组成部分的疾病评估和管理。
Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (beta-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17-31% and 16-28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.