| Abstract | Cyanobacteria colonize different environments and blooms can occur in both contaminated and non-contaminated water bodies (freshwater, brackish and marine areas). Among 150 known cyanobacteria genera, more than 40 species are able to produce toxins, which are natural compounds that differ from both a chemical and toxicological point of view and are responsible for acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin-producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non-toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH (Fluorescence In Situ Hybridization) analysis to detect Microcystis aeruginosa at the species level even at relatively low concentrations in freshwater. The FISH probe, MicAerD03, was designed using the ARB software with the Silva database within the framework of the MicroCoKit project, also with the intention of adding it to the microarray from the EU project, uAQUA, for freshwater pathogens, which had only genus level probes for Microcystis.
We tested various fixative methods to minimise the natural autofluorescence from chlorophyll-a and certain accessory pigments (viz., phycobilins and carotenoids). The FISH probe was tested on pure cultures of Microcystis aeruginosa, and then successfully applied to water samples collected from different sampling points of the Tiber River (Italy), using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5' end with horse-radish peroxidase (HRP-MicAerD03) to apply the CAtalysed Reported Deposition-FISH (CARD-FISH) for increasing the fluorescence signal of the mono-fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the EU MicroCokit project indicated that microarray signals for Microcystis were coming from single cells and not colonial cells. We confirmed this with the CARD-FISH protocol used here to validate the microarray signals for Microcystis detected at the genus level in MicroCokit.
This paper provides a new early warning tool for investigating M. aeruginosa at the species level even at low cell concentrations in surface water, which can be added to the uAqua microarray for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa |
| Authors | Barra Caracciolo A, Dejana L, Fajardo C, Grenni P, Martin M, Mengs G, Sánchez-Fortún S, Lettieri T, Saccà ML, Medlin L |
| Text | 400042 2019 10.1016/j.microc.2019.05.017 Scopus scopus Cyanobacteria FISH probes CARD FISH Algal bloom River water Early warning tool A new fluorescent oligonucleotide probe for in situ identification of Microcystis Aeruginosa in freshwater Barra Caracciolo A, Dejana L, Fajardo C, Grenni P, Martin M, Mengs G, Sanchez Fortun S, Lettieri T, Sacca ML, Medlin L Barra Caracciolo A, Dejana L, Grenni P, Sacca ML IRSA Fajardo C,, Martin M, Sanchez Fortun S Facultad de Veterinaria, Complutense University Avenida Puerta de Hierro s/n, 28040 Madrid, Spain. Mengs G, Natural Biotec S.L., Parque Cientifico de Madrid, Ctra. de Colmenar Viejo, km 15. Cantoblanco, Pabellon C, 28049 Madrid, Spain. Lettieri T European Commission, Joint Research Centre JRC , Directorate D, Sustainable Resources, Water and Marine Resources Via E. Fermi, 2749, I 21027 Ispra VA , Italy. Sacca ML, Medlin L Marine Biological Association of the UK, The Citadel, Plymouth PL1 2PB, UK Cyanobacteria colonize different environments and blooms can occur in both contaminated and non contaminated water bodies freshwater, brackish and marine areas . Among 150 known cyanobacteria genera, more than 40 species are able to produce toxins, which are natural compounds that differ from both a chemical and toxicological point of view and are responsible for acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH Fluorescence In Situ Hybridization analysis to detect Microcystis aeruginosa at the species level even at relatively low concentrations in freshwater. The FISH probe, MicAerD03, was designed using the ARB software with the Silva database within the framework of the MicroCoKit project, also with the intention of adding it to the microarray from the EU project, uAQUA, for freshwater pathogens, which had only genus level probes for Microcystis. We tested various fixative methods to minimise the natural autofluorescence from chlorophyll a and certain accessory pigments viz., phycobilins and carotenoids . The FISH probe was tested on pure cultures of Microcystis aeruginosa, and then successfully applied to water samples collected from different sampling points of the Tiber River Italy , using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5 end with horse radish peroxidase HRP MicAerD03 to apply the CAtalysed Reported Deposition FISH CARD FISH for increasing the fluorescence signal of the mono fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the EU MicroCokit project indicated that microarray signals for Microcystis were coming from single cells and not colonial cells. We confirmed this with the CARD FISH protocol used here to validate the microarray signals for Microcystis detected at the genus level in MicroCokit. This paper provides a new early warning tool for investigating M. aeruginosa at the species level even at low cell concentrations in surface water, which can be added to the uAqua microarray for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa 148 Published version 09/03/2019 A new fluorescent oligonucleotide probe for in situ identification of Microcystis aeruginosa in freshwater Messa a punto di una sonda FISH per la rapida identificazione del cianobatterio M. aeruginosa in acque superficiali MicrochemMicrocystisaeruginosa2019.pdf Articolo in rivista Academic Press etc. 0026 265X Microchemical journal Print Microchemical journal Print Microchemical journal. Print DEJANA LAURA anna.barracaracciolo BARRA CARACCIOLO ANNA paola.grenni GRENNI PAOLA MICROCOKIT Microbial Community based sequencing analysis linked to anthropogenic pressures MicroCoKit to address the water quality |
