title: Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria.
abstract: The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.
authors.affiliation: Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Milan, Italy.
authors.forename: Bianca
authors.initials: B
authors.lastname: Castiglioni
authors.forename: Ermanno
authors.initials: E
authors.lastname: Rizzi
authors.forename: Andrea
authors.initials: A
authors.lastname: Frosini
authors.forename: Kaarina
authors.initials: K
authors.lastname: Sivonen
authors.forename: Pirjo
authors.initials: P
authors.lastname: Rajaniemi
authors.forename: Anne
authors.initials: A
authors.lastname: Rantala
authors.forename: Maria Angela
authors.initials: MA
authors.lastname: Mugnai
authors.forename: Stefano
authors.initials: S
authors.lastname: Ventura
authors.forename: Annick
authors.initials: A
authors.lastname: Wilmotte
authors.forename: Christophe
authors.initials: C
authors.lastname: Boutte
authors.forename: Stana
authors.initials: S
authors.lastname: Grubisic
authors.forename: Pierre
authors.initials: P
authors.lastname: Balthasart
authors.forename: Clarissa
authors.initials: C
authors.lastname: Consolandi
authors.forename: Roberta
authors.initials: R
authors.lastname: Bordoni
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mezzelani
authors.forename: Cristina
authors.initials: C
authors.lastname: Battaglia
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
mesh_terms: D015373:Bacterial Typing Techniques; D000458:Cyanobacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D005618:Fresh Water / Q000382:microbiology*; D014644:Genetic Variation; D008025:Ligases / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011110:Polymorphism, Genetic; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013045:Species Specificity
chemical_list: D015342:DNA Probes; D012336:RNA, Ribosomal, 16S; D008025:Ligases