柑橘黄龙病菌双重实时荧光PCR检测方法的建立  

Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’

作　　者：李镇希 潘睿翾 许美容[1] 郑正[1] 邓晓玲[1] LI Zhenxi;PAN Ruixuan;XU Meirong;ZHENG Zheng;DENG Xiaoling(Citrus Huanglongbing Reseach Laboratory,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学柑橘黄龙病研究室,广州510642

出　　处：《园艺学报》2023年第1期188-196,共9页Acta Horticulturae Sinica

基　　金：广东省重点领域研发计划项目(2019B020217003);广西创新驱动发展专项(桂科AA18118046)。

摘　　要：建立基于双重荧光定量PCR检测柑橘黄龙病菌(Candidatus Liberibacter asiaticus,CLas)的方法,并对其准确性进行验证。通过受试者工作特征(ROC)曲线、净重分类改善指数(NRI)和综合判别改善指数(IDI)分析7对CLas实时荧光PCR引物及其联合检测的准确性,评价RNRf/RNRr联合CLas-4G/HLBr的双重实时荧光PCR对CLas的检测价值。ROC曲线分析结果显示,表现最好的引物是RNRf/RNRr,其曲线下面积(AUC)为0.971,高于其他6对引物;其次是CLas-4G/HLBr引物,AUC为0.966;在双引物联合检测中,RNRf/RNRr+CLas-4G/HLBr引物串联检测、CQULA04F/CQULA04R+RNRf/RNRr引物并联检测的相关性能指标较优,AUC分别为0.963和0.966,并且RNRf/RNRr+CLas-4G/HLBr引物串联检测同时具备较高的敏感性(85.19%)和特异性(99.25%)。NRI分析结果表明RNRf/RNRr+CLas-4G/HLBr串联检测的诊断能力相较于LJ900f/LJ900r的单一引物实时荧光PCR显著提升了6.00%(P<0.05)。使用RNRf/RNRr和CLas-4G/HLBr两对引物进行双重实时荧光PCR,发现RNRf/RNRr+CLas-4G/HLBr双重实时荧光PCR比单一引物实时荧光PCR有更高的灵敏度。因此,RNRf/RNRr+CLas-4G/HLBr双重实时荧光PCR可作为柑橘黄龙病低发区的早期诊断工具。This study aimed to establish a duplex real-time PCR assay to detect‘Candidatus Liberibacter asiaticus’(CLas).Receiver operating characteristic(ROC)curve,net reclassification improvement(NRI),and integrated discrimination improvement(IDI)were used to analyze the accuracy of seven real-time PCR primer sets and their pair-wise combination detections,and evaluate the diagnostic value of duplex real-time PCR with primer sets RNRf/RNRr and CLas-4G/HLBr.The best diagnostic performance in the single primer assay was RNRf/RNRr with an area under the curve(AUC)of 0.971,followed by CLas-4G/HLBr with an AUC of 0.966.In combined real-time PCR detection by two primer sets,both the tandem use of primer set RNRf/RNRr+CLas-4G/HLBr and the parallel use of primer set CQULA04F/CQULA04R+RNRf/RNRr showed good diagnostic performances with AUCs of 0.963 and 0.966,and the RNRf/RNRr+CLas-4G/HLBr tandem assay had both high(85.19%)and specificity(99.25%).NRI analysis showed that the predictive ability of the tandem use of primer set RNRf/RNRr+CLas-4G/HLBr was significantly increased by 6.00%compared with the single use of primer set LJ900f/LJ900r(P<0.05).Using RNRf/RNRr and CLas-4G/HLBr primer sets to perform duplex real-time PCR,it was found that RNRf/RNRr+CLas-4G/HLBr duplex real-time PCR was more sensitive than single primer real-time PCR.Therefore,RNRf/RNRr+CLas-4G/HLBr duplex real-time PCR could be used as an early diagnostic tool in low incidence areas of citrus Huanglongbing.

关 键 词：柑橘黄龙病 实时荧光定量PCR 引物 联合诊断 

分 类 号：S666[农业科学—果树学]