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%), R-2 of the standard curve (0.999), the limit of detection (LOD, 5.7 fg mu L-1), and the limit of quantification (LOQ, 1.86 x 10(4) gene copies mu L-1). Further, the method was verified by the correlation between the mic gene abundance and MIB concentration 50 field samples from different reservoirs (R-2 = 0.614, p < 0.001) and one reservoir (R-2 = 0.752, p < 0.001), suggesting its potential as an alternative warning tool to evaluate the risk of MIB problems in source water.

Automated summary

A novel RT-qPCR method based on detecting the MIB synthesis gene was proposed to evaluate the MIB-producing potential in source water, showing a wider coverage than previous primer sets. The method was efficient, with proven amplification efficiency, LOD, and LOQ, and was further verified in field samples, suggesting its potential as an alternative warning tool for assessing MIB problems in source water.