Conditions for loop-mediated isothermal amplification (LAMP) and a nonmacerating DNA extraction method to assay for huanglongbing (citrus greening) disease
2008
Okuda, M.(National Agricultural Research Center for Kyushu Okinawa Region, Koshi, Kumamoto (Japan)) | Kawano, S. | Murayama, Y. | Iwanami, T.
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 rplKJAL-rpoB 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 61degC to 65degC. 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 reagents.
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