Huanglongbing (HLB) is a citrus infectious disease caused by Candidatus Liberibacters 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 (STD-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/test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that 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, and quantitative screening method for accurately controlling HLB and a universal method for establishing PCR assay for various pathogens.
Keywords: CLas; Huanglongbing; citrus; duplex PCR; internal standard; mutant primers.