An Inhibitor-Monitorable Single-Tube Duplex Quantitative Real-Time PCR Assay for the Detection of ‘Candidatus Liberibacter asiaticus’

    Authors and Affiliations
    • Weida Huang1
    • Zecheng Zhong1
    • Zhihua Lin2
    • Jinlian Zhang3
    • Jinhua Liu4
    • Tingsu Chen3
    • Tingdong Li1 5
    • Shiyin Zhang1 5
    • Shengxiang Ge1 5
    1. 1National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, School of Public Health, Xiamen University, Xiamen, Fujian 361102, China
    2. 2Integrated Technical Service Center, Zhangzhou Customs, Zhangzhou, Fujian 363000, China
    3. 3Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China
    4. 4Zhejiang Yang Sheng Tang Institute of Natural Medicine Co., Ltd., Hangzhou, Zhejiang 310000, China
    5. 5State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen, Fujian 361102, China

    Published Online:https://doi.org/10.1094/PDIS-09-22-2168-SR

    Huanglongbing (HLB) is a citrus infectious disease caused by ‘Candidatus Liberibacter’ spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3′-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of ‘Candidatus Liberibacter asiaticus’ (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

    Literature Cited