柑桔黄龙病的常规PCR及荧光定量PCR检测

Detection of Citrus Huanglongbing by Conventional and Two Fluorescence Quantitative PCR Assays

目的为柑桔黄龙病的早期诊断和寄主体内病原菌的动态监测提供一种稳定、可靠的检验检疫技术。方法利用亚洲韧皮杆菌核糖体蛋白基因rplJ/rplL设计了2对PCR引物CQULA03F/CQULA03R、CQULA04F/CQULA04R和1条TaqMan探针CQULAP1,以此为基础建立了常规PCR和TaqMan探针法、SYBRGreenI荧光染料法两种荧光定量PCR(共3种)反应体系;确定了3种体系各自的检测灵敏度、特异性和准确性,据此对3种体系进行了比较;从2004年7月到2005年5月还利用常规PCR和TaqMan探针荧光定量PCR体系完成了对柑桔黄龙病病原菌在寄主体内的周年变化的动态监测。结果两种荧光定量PCR方法的灵敏度比常规PCR高出至少2～3个数量级,而TaqMan探针法由于使用了杂交探针,其特异性尤其可靠,另外两种定量PCR较小的产物片段使得它们具有更好的稳定性,加上荧光定量PCR方法本身受污染可能性小、操作简便等固有优势,使得前者更适合于柑桔黄龙病的检测。结论本研究建立的2种荧光定量PCR方法可以为柑桔黄龙病的病害早期诊断以及柑桔黄龙病病原菌近缘种的甄别提供准确、灵敏、快速的检验检疫技术。

[Objective] The project aimed to develop an accurate, sensitive and fast technique for the early diagnosis of Huanglongbing and identification of Ca. L. asiaticus. [Method] Conventional and two types of Fluorescence Quantitative Polymerase chain reaction （PCR） including SYBR Green I DNA-binding dye and TaqMan Probe approaches were established and optimized based on two pair of primers, CQULA03F/CQULA03R, CQULA04F/CQULA04R. TaqMan fluorescent probes were designed from the ribosomal protein gene sequence rplJ/rplL of Candidatus Liberibacter asiaticus. The specificity and limitation of both SGI and TaqMan FQ -PCR methods were compared with conventional PCR. This was the PCR for suspension detection of Ca. L. asiaticus collection from citrus leave midrib and recombinant plasmid DNA with a ribosomal protein gene target fragment. We also use the detection systems to monitor the dynamic change of the Huanglongbing pathogen in the host from July of 2004 to May of 2005 and obtained some useful information about the interaction between the host and the pathogen. [Result] The three techniques （Conventional-PCR, SYBR Green I and TaqMan Q-PCR） stably detected target bacteria from symptomatic or asymptomatic citrus samples, and the sensitivity of FQ-PCR is higher than Conventional-PCR at 100-1000-fold （these are 439.0 fg·μl^-1, 4.39 fg·μl^-1, 0.44 fg·μl^-1 for conventional PCR , TaqMan and SGI PCR respectively）; the TaqMan Q-PCR has robust specificity and higher accuracy but is more expensive compared with the SGI approach. [Conclusion] This specific, accurate, sensitive and fast technique will play a key role for the early diagnosis of Huanglongbing and identification of Ca. L. asiaticus.