A qPCR method for detection of 'Candidatus Liberibacter asiaticus': Optimization of selected assay parameters

'Candidatus Liberibacter asiaticus' (CLas), the presumed causative agent of Huanglongbing (HLB, citrus greening), threatens citrus production worldwide. With no cure available, infected trees decline rapidly and entire groves can be lost. Slowing the spread of HLB depends on early, accurate detection of CLas and prompt removal of infected trees. In California, quantitative polymerase chain reaction (qPCR) remains the gold standard test. Reaching the theoretical detection limit-detecting a single CLas cell in a sample-requires tuning the full workflow, not just the primers and probe. Here, we tested four practical levers: PCR plate color (clear, black, and white); pipetting consistency; reported PCR enhancers; and the addition of a reverse transcription step to enable RT-qPCR on total nucleic acid extracts. White plates increased fluorescence signal and tightened Ct values across replicates. Improved pipetting reduced operator-dependent variability. Among the tested enhancers, 1-2% glycerol improved detection at low CLas titer levels. Adding reverse transcription lowered Ct values by at least 5 cycles, improving detection by at least ∼32-fold. Together, these low-cost changes push routine qPCR toward single-bacterium detection while reducing variability in CLas surveillance.