NEW MOLECULAR TOOLS: APPLICATION OF THE µAQUA PHYLOCHIP AND CONCOMITANT FISH PROBES TO STUDY FRESHWATER PATHOGENS FROM SAMPLES TAKEN ALONG THE TIBER RIVER, ITALY
2017
Current knowledge about aquatic pathogens are scarce because bacteria, protozoans, algae and their
toxins occur at low concentrations, making them difficult to measure directly or to filter sufficient
volumes to facilitate detection. We developed and validated tools to detect pathogens in freshwater
systems. To evaluate impacts on water quality, a phylogenetic microarray was developed in the EU
project μAQUA to detect simultaneously numerous pathogens and was applied in MicroCoKit, to
samples taken from four locations from two seasons for two years along the length of the Tiber River,
Italy. The sites ranged from a pristine site near its source to ones contaminated by agricultural, industrial
and anthropogenic waste moving downstream to near its mouth. Fifty litres were collected and
concentrated using a hollow-fibre ultrafiltration, a rapid method with minimal cell loss to provide a
concentrate for downstream analysis. The 60 Da cut-off ensures many organics, such as toxins, will be
concentrated for analysis. Aliquots from the concentrate were preserved in TRI-Reagent and total RNA
extracted, labelled and hybridised to the phylochip to detect pathogenic bacteria, protozoa and toxic
cyanobacteria. The microarray results gave positive signals for all pathogens. Calibration curves
enabled us to infer cell concentrations. Cross validation was performed using FISH probes for selected
toxic cyanobacteria and hybridised to aliquots taken from the raw water prior to concentration by the
hollow fibre filters.
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