Abstract: The presence of high concentrations of polysaccharides，polyphenols，pigment，and other secondary metabolites in mature citrus leaves is big challenging to obtain high quality DNA/RNA for citrus disease research. In this study，a reliable，fast and enhanced total nucleic acid extraction protocol（CTAB-Triton），modified from the cetyltrimethylammonium bromide（CTAB）method，was established for DNA extraction of mature citrus tissues. The novel extraction buffer was superior to traditional buffer in cell lysis，and avoided low yield due to CTAB-DNA co-precipitation when temperature was lower than 15 ℃. Meanwhile，good-quality DNA and higher DNA yield were obtained through this extraction method，comparing with the commercial DNA isolation kits. The infection rate of‘Candidatus Liberibacter asiaticus’（CLas）extracted by CTAB-Triton and Sephadex G-50 extraction method was compared，CTAB-Triton method significantly improved the detection rate. SYBR Green-based quantitative PCR（qPCR）assay was performed to compare the concentration of CLas within different parts of pummelo leaves. This new method was also suitable for the extraction of citrus fungi，bacteria，and DNA/RNA viral diseases for the pathogen detection and genetic diversity analysis.

Key words: citrus, ‘Candidatus Liberibacter asiaticus’, huanglongbing, DNA extraction, citrus disease detection