摘 要 ：根据地毯草黄单孢Xac306菌株(Xanthomonas axonopodis pv.citri, Xac 306)已知全基因组中独有蛋白基因序列设计的特异性引物对和探针,建立并优化SYBR Green I (SGI) 荧光染料和TaqMan探针实时荧光定量PCR检测体系,用于柑桔溃疡病早期诊断鉴定。结果表明,建立的两种定量PCR体系均能特异地检出Xac的细胞和其基因组DNA,而对其它测试的植物病原菌和柑桔表面的腐生黄单孢菌都不能检出。SGI法和TaqMan探针法对Xac细菌悬浮液的检测灵敏度均可达到1~5个细菌/反应,对Xac靶标片段DNA的检测灵敏度可达1fg/μL。两种定量PCR检测方法比常规PCR灵敏度高2~3个数量级。对田间采集的328个柑桔显症、疑似症状和无症带菌材料富集培养样品进行了实际检测,结果表明,实时荧光PCR适合柑桔无症带菌样品的早期检测。
Abstract:The primers and probes were designed based on the putative protein gene sequence in complete genome of Xanthomonas axonopodis pv.citri strain 306 (Xac 306), and used separately to establish and optimize SYBR Green I dye (SGI) and TaqMan probe real time fluorescent quantitative PCR (RTi-PCR) approaches for citrus bacterial canker disease detection. The results showed that only the target pathogen Xac and its DNA can be detected, while other bacteria including saprophytic xanthomonads from citrus leaf surface and related plant pathogens cannot be detected. The sensitivity of real time fluorescent quantitative PCR is higher than that of conventional PCR by 100-1000 fold. The practice detections of suspicious symptomatic or asymptomatic samples collected in citrus orchards from citrus diseased area have been performed with fluorescent RTi-PCR method after pathogen enrichment. The results suggested that, as a sensitive and reliable diagnosis and identification method, RTi-PCR meets the requirement of pre-symptomatic detection of citrus pathogen.