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

文献类型： 外文期刊

作者： Huang, Weida 1 ; Zhong, Zecheng 1 ; Lin, Zhihua 2 ; Zhang, Jinlian 3 ; Liu, Jinhua 4 ; Chen, Tingsu 3 ; Li, Tingdong 1 ; Zhang, Shiyin 1 ; Ge, Shengxiang 1 ;

作者机构： 1.Xiamen Univ, Natl Inst Diagnost & Vaccine Dev Infect Dis, Chinese Acad Med Sci, NMPA Key Lab Res & Evaluat Infect Dis Diagnost Tec, Xiamen 361102, Fujian, Peoples R China

2.Zhangzhou Customs, Integrated Tech Serv Ctr, Zhangzhou 363000, Fujian, Peoples R China

3.Guangxi Acad Agr Sci, Microbiol Res Inst, Nanning 530007, Guangxi, Peoples R China

4.Zhejiang Yang Sheng Tang Inst Nat Med Co Ltd, Hangzhou 310000, Zhejiang, Peoples R China

5.Xiamen Univ, Sch Publ Hlth, Dept Lab Med, State Key Lab Vaccines Infect Dis,Xiang An Biomed, Xiamen 361102, Fujian, Peoples R China

关键词： citrus; CLas; duplex PCR; huanglongbing; internal standard; mutant primers

期刊名称：PLANT DISEASE （ 影响因子：4.5； 五年影响因子：5.0 ）

ISSN： 0191-2917

年卷期： 2024 年 108 卷 5 期

页码：

收录情况： SCI

摘要： 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.

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