Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa
Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa
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DOI：
10.1007/s10658-006-9009-4
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发表时间：
2006-06-01
影响因子：
1.8
通讯作者：
Civerolo, Edwin L.
中科院分区：
文献类型：
--
作者：
Francis, Marta;Lin, Hong;Civerolo, Edwin L.
苛养木杆菌是许多经济作物的重要病原菌。X的检测。fastidiosa是困难的，因为昆虫和无症状植物组织中的细菌浓度低，并且在受感染的植物中分布不均匀。开发了一种双重用途的常规PCR和定量PCR（TaqMan（TM））系统用于X的通用检测。苛养菌株。引物HL 5和HL 6，设计用于扩增四个Xylella菌株的测序基因组共有的独特区域，从与葡萄皮尔斯病、杏仁叶焦枯和夹竹桃叶焦枯病相关的菌株和来自与柑橘杂色失绿相关的Xf菌株的DNA扩增了221 bp片段。使用在水和葡萄提取物中每个反应5 - 10（5）个细胞和在昆虫DNA中10-10（5）个细胞的Xylella浓度获得标准曲线。回归曲线相似，相关系数r（2）> 0.97。在定量PCR中，C-t值范围在20和36个循环之间，每个反应5-10（5）个细菌细胞。没有获得扩增子与几个非Xf细菌菌株测试，包括相关的植物病原体，葡萄内生细菌和内共生细菌分离的玻璃翅神枪手。该方法可用于葡萄、杏仁和媒介昆虫中Xf的临床诊断。所描述的程序是可靠的检测的病原体具有高度的灵敏度和特异性。
Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan (TM)) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce's disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10(5) cells per reaction in water and grape extracts and 10-10(5)cells in insect DNA. Regression curves were similar, with correlation coefficients of r(2) > 0.97. In quantitative PCR, C-t values ranged between 20 and 36 cycles for 5-10(5) bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.