Development of a Real-time Fluorescence Loop-mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

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• 作者：Weigang Kuang ; Laixin Luo ; Wenna Gao ; Yahong Lei ; Qingyang lv and Jianqiang Li
• 刊名：Journal of Phytopathology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：165
• 期：2
• 页码：82-90
• 全文大小：804K
• ISSN：1439-0434

文摘

Burkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop-mediated isothermal amplification (LAMP) assay with a real-time fluorometer was developed. The analysis of 16S-23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real-time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real-time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.