关键词: Cadidatus Liberibacter asiaticus, 黄龙病, SYBR Green I, TaqMan, 荧光定量PCR

Abstract: Two types of Fluorescence Quantity Polymerase chain reaction (PCR) including SYBR Green I DNA-binding dye and TaqMan Probe approaches are established and optimized based on two pair of primers, CQULA03F/CQULA03R, CQULA04F/CQULA04R and TaqMan fluorescent probe designed from ribosomal protein gene sequence of Ca. L. asiaticus. The specificity and limitation of both SGI and TaqMan FQ -PCR methods are compared with classical PCR for detecting suspensions of felted collection of Ca. L. asiaticus from midrib of citrus leaves and recombinant plasmid DNA with target fragment of ribosomal protein gene. The three techniques (Classical-PCR, SYBR Green I and TaqMan Q-PCR) can stably detect target bacteria from symptomatic or asymptomatic citrus samples, and the sensitivity of FQ-PCR is higher than Classical-PCR at 100～1000-fold (these are 439.0 fg/μl, 4.39 fg/μl, 0.44 fg/μl for classical PCR ,TaqMan and SGI PCR respectively)；the TaqMan Q-PCR has robust specificity and higher accuracy but is more expensive comparing with SGI approach. This specific, accurate, sensitive and fast technique will play a key role for the early diagnosis of HLB and identification of Ca. L. asiaticus.

Key words: Cadidatus Liberibacter asiaticus, HLB, SYBR Green I, TaqMan, FQ-PCR