Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (1): 188-196.doi: 10.16420/j.issn.0513-353x.2021-0502

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

LI Zhenxi, PAN Ruixuan, XU Meirong, ZHENG Zheng, DENG Xiaoling*()   

  1. Citrus Huanglongbing Reseach Laboratory,South China Agricultural University,Guangzhou 510642,China
  • Received:2022-04-25 Revised:2022-06-17 Online:2023-01-25 Published:2023-01-18
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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.

Key words: citrus Huanglongbing, real-time PCR, primer, combined diagnoses

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