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期 刊 ：植物保护学报 2012年

关键词：柑橘黄龙病；常规PCR；巢式PCR；SYBR GreenⅠ荧光定量PCR；

Keywords:citrus huanglongbing, conventional PCR, nested-PCR, SYBR GreenⅠ-qPCR,

摘 要 ：为了评价3种PCR分子检测体系对柑橘黄龙病(citrus huanglongbing,HLB)大田诊断效果,综合比较了常规PCR、巢式PCR和SYBR GreenⅠ荧光定量PCR(SGⅠ-qPCR)方法对柑橘黄龙病菌检测的灵敏度、特异性和准确度等参数,并用SYBR GreenⅠ荧光定量PCR和巢式PCR监测广西柑橘园疑似HLB样品425个,比较了2种检测体系的阳性检出率。基于CQULA04F/CQULA04R引物对的SYBR GreenⅠ荧光定量PCR的灵敏度可达10 ag/μL;而巢式PCR灵敏度为100 ag/μL,巢式PCR较常规PCR检测灵敏度高10⁴倍。疑似样品 HLB病原SYBR GreenⅠ荧光定量PCR和巢式PCR检出率分别为46.6%、40.0%。各检测体系的灵敏性、特异性、符合度依次为SYBR GreenⅠ荧光定量PCR>巢式PCR>常规PCR。研究表明,SYBR GreenⅠ荧光定量PCR可作为果园大规模HLB早期诊断和监测的首选,而在缺乏定量检测仪器时,巢式PCR也可用于HLB的检测,但需注意避免空气污染导致的假阳性。

Abstract:In order to provide practical detection approach for early diagnosis and dynamic monitoring of citrus huanglongbing (HLB) pathogen. The sensitivity, specificity and accuracy of 3 type PCR approaches were compared. Total 425 suspected citrus samples with or without symptom of HLB from plantation of Hepu County,Guangxi were amplified and confirmed by nested-PCR and SYBR GreenⅠ-qPCR (SGⅠ-qPCR). The effectiveness and the positive rates of the two PCR were evaluated. The results showed that the detection sensitivity of SGⅠ-qPCR for HLB pathogen DNA was 10 ag/μL, while was 100 ag/μL for nested-PCR. Conventional PCR detection sensitivity was 10⁴ times lower than nested-PCR method. Positive rate percent of detection for suspected citrus samples were in 46.6% and 40.0% by SGⅠ-qPCR and nested-PCR, respectively, which indicated that SGⅠ-qPCR was able to provide more accurate diagnoses than nested-PCR. The sensitivity and veracity of conventional PCR, nested-PCR and SGⅠ-qPCR system were enhancing in turn. Practically, SGⅠ-qPCR should be concerned priority selection in large scale early diagnosis and dynamic monitoring in citrus plantation. As the PCR instrument of quantitative detection not available, nested-PCR could be used for better detection of HLB as long as well controlling of the air pollution of amplified procedure, in order to prevent the false positive amplification.

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