LAMP法を用いたカンキツグリーニング病原細菌検出の反応条件と非磨砕DNA抽出法の検討

抄録

A loop-mediated isothermal amplification (LAMP) assay to detect the causal organism of huanglongbing (citrus greening) was developed. Primers were designed from the nucleotide sequence of the rpl KJAL-rpo B operon of Candidatus Liberibacter asiaticus. DNA amplification was greatly accelerated when loop primers were added to the reaction, and the reaction kinetics was correlated with the reaction temperature from 61°C to 65°C. Sodium hydroxide was used in a rapid DNA extraction method (called alkali extraction) that does not require maceration of leaves, and specific amplifications in the LAMP reaction were obtained from all samples prepared from Ca. L. asiaticus-infected plants. With these methods, Ca. L. asiaticus was detected when one infected leaf was mixed with four healthy ones. The threshold times for turbidity were equal to those for samples prepared from the same leaves using commercially produced regents.