Development and field assessment of a quantitative PCR for the detection and enumeration of the noxious bloom-former Anabaena planktonica

Abstract

Anabaena planktonica is a harmful, bloom-forming freshwater cyanobacterium, which has arrived recently in New Zealand. In the short time since its incursion (<10 yr), A. planktonica has spread rapidly throughout lakes in the North Island. To date, the identification and enumeration of A. planktonica has been undertaken using light microscopy. There is an urgent demand for a highly sensitive and specific quantitative detection method that can be combined with a high sample processing capability in order to increase sampling frequency. In this study, we sequenced 36 cyanobacterial 16S rRNA genes (partial), complete intergenic transcribed spacers (ITS), and 23S rRNA genes (partial) of fresh-water cyanobacteria found in New Zealand. The sequences were used to develop an A. Planktonica specific TaqMan QPCR assay targeting the long ITS1-L and the 5´ terminus of the 23S rRNA gene. The QPCR method was linear (R2 = 0.999) over seven orders of magnitude with a lower end sensitivity of approximately five A. planktonica cells in the presence of exogenous DNA. The quantitative PCR (QPCR) method was used to assess the spatial distribution and seasonal population dynamics of A. planktonica from the Lower Karori Reservoir (Wellington, New Zealand) over a five-month period. The QPCR results were compared directly to microscopic cell counts and found to correlate significantly (95% confidence level) under both bloom and non-bloom conditions. The current QPCR assay will be an invaluable tool for routine monitoring programs and in research investigating environmental factors that regulate the population dynamics and the blooming of A. planktonica.