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.

Potentials and limitations of molecular diagnostic methods in food safety

“…First, the LDR-UA technology has been used for the detection and classification of water borne pathogens such as cyanobacteria [6]. The authors used a mix of 19 probe sets able to detect over 300 cyanobacteria strains and classify them in 14 phylogenetic lineages.…”

“…The LDR-UA has proved to be very sensitive, being able to detect target PCR product up to 50 pM, a concentration certainly not visible with an EtBr agarose gel or with a conventional microarray [6]. The high sensitivity of LDR-UA is mainly due to the linear amplification performed during the reaction and to the concentration of the fluorescent probes on the UA spot.…”

Molecular genetic tools for environmental monitoring of New Zealand's aquatic habitats, past, present and the future

“…For example, Gescher et al (2008) and Ki and Han (2006) have developed microarrays for toxic marine phytoplankton and Castiglioni et al (2004) for cyanobacteria. Microarrays are limited to detection of the targets that they are designed for and we anticipate that for environmental monitoring purposes they will be superseded by NGS.…”

Molecular biology techniques and applications for ocean sensing

“…The small subunit ribosomal RNA (SSU rRNA) gene sequence has been used to develop phylogenetic microarrays with the goal of detecting microbes representing different phylogenetic groups in the environment. Phylogenetic microarrays have been developed for terrestrial environments (Small et al, 2001;DeSantis et al, 2007), all known sulphate reducers (Loy et al, 2002), and aquatic cyanobacteria (Castiglioni et al, 2004). An electronic microarray (NanoChip) using the SSU rRNA gene sequence for detection of bacteria associated with coastal harmful algal blooms was developed (Barlaan et al, 2007).…”