|Zheng, Zheng - South China Agricultural University|
|Deng, Xiaoling - South China Agricultural University|
Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/1/2015
Publication Date: 11/1/2015
Citation: Zheng, Z., Deng, X., Chen, J. 2015. Enhancing detection sensitivity and accuracy on “Candidatus Liberibacter asiaticus” through next generation sequencing technology. American Phytopathological Society Annual Meeting. 105:S4.159.
Technical Abstract: “Candidatus Liberibacter asiaticus” is associated with citrus Huanglongbing (HLB, yellow shoot disease). Detection of this unculturable bacterium exclusively depends on polymerase chain reaction (PCR). Appropriate primer design is key to assure detection sensitivity and accuracy, which depend on quality of available bacterial sequences. Next Generation Sequencing (NGS) is a term describing several recent sequencing technologies with high throughput capacity. For example, the Illumina MiSeq platform can generate a Giga-base level sequence data set in one instrumental run. Therefore, NGS increases nucleotide coverage (high sequence accuracy) and allows thorough collection of all DNA species (deep sequencing) in a sample. This study utilized the Illumina MiSeq data sets from three HLB samples (Guangdong of China, California, and Florida) to enhance “Ca. L. asiaticus” detection. Three whole genome sequences of “Ca. L. asiaticus” strains were assembled. Genome comparison revealed the presence of a gene with a copy number of five. Primer set designed from this gene significantly increased PCR detection sensitivity over the standard 16S rDNA-based primer set. The 16S rRNA gene from whole genome sequences were compared with those previously published. A missing G base was found in the published signature sequence of “Ca. L. asiaticus” and a new primer set was designed to correct the deficiency. Analyses on the MiSeq sequence data from California clarified possible presence of “Ca. L. africanus” by revealing a previous error in primer design. In summary, NGS technology has a high potential for enhancing HLB diagnosis.