三种分子检测体系的比较及柑橘果园黄龙病监测

Comparative of citrus huanglongbing molecular detection approaches and monitoring of the orchards

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 Green Ⅰ荧光定量PCR(SG Ⅰ-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 Green Ⅰ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 Green Ⅰ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。疑似样品的HLB病原SYBR Green Ⅰ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SYBRGreen Ⅰ荧光定量PCR>巢式PCR>常规PCR。研究表明,SYBR Green Ⅰ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。

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 approa- ches 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 Green I -qPCR （ SG I -qPCR）. The effectiveness and the positive rates of the two PCR were evaluated. The results showed that the detection sensitivity of SG I -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 SG I -qPCR and nested-PCR, respectively, which indicated that SG I -qPCR was able to provide more accurate diag- noses than nested-PCR. The sensitivity and veracity of conventional PCR, nested-PCR and SG I -qPCR system were enhancing in turn. Practically, SG I -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 controllingof the air pollution of amplified procedure, in order to prevent the false positive amplification.