Evaluation of the MIB-producing potential based on real-time qPCR in drinking water reservoirs

Cyanobacteria release 2-methylisoborneol (MIB) as a secondary metabolite. Here, we propose a reverse transcription quantitative real-time PCR (RT-qPCR) based method to evaluate the MIB-producing potential in source water by detecting the MIB-synthesis gene (mic). A MIBQSF/R primer set was designed based on 35 mic gene sequences obtained from 12 pure-cultured MIB-producing strains and 23 sequences from the NCBI database. This primer set successfully identified all known 43 MIB-producing cyanobacterial strains (12 from this study and 31 from the NCBI database), belonging to different genera, showing a wider coverage than previous primer sets. The efficiency of the method was proved by the amplification efficiency (E = 91.23%), R² of the standard curve (0.999), the limit of detection (LOD, 5.7 fg μL^-1), and the limit of quantification (LOQ, 1.86 × 10⁴ gene copies μL^-1). Further, the method was verified by the correlation between the mic gene abundance and MIB concentration 50 field samples from different reservoirs (R² = 0.614, p < 0.001) and one reservoir (R² = 0.752, p < 0.001), suggesting its potential as an alternative warning tool to evaluate the risk of MIB problems in source water.