METHODS Typing of toxinogenic Microcystis from environmental samples by multiplex PCR Youness Ouahid & Francisca Fernández del Campo Received: 19 June 2009 / Revised: 4 September 2009 / Accepted: 6 September 2009 / Published online: 3 October 2009 # Springer-Verlag 2009 Abstract Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non- ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC- producing Microcystis in field samples, even scattered colonies. Keywords mcy genes . NRPS . PKS . Microcystis . Multiplex PCR . Bloom biomass Introduction Toxic cyanobacterial blooms are potential health hazards in water supply reservoirs, Microcystis, Planktothrix, Anabaena and Aphanizomenon being the most usually present genera. A bloom can consist of one or various species with one or a mixture of toxic and non-toxic strains. Microcystis is probably the most world-abundant toxic cyanobacterial genus, the majority of its strains producing microcystins (MCs). MCs are hepatotoxins that specifically inhibit serine/ threonine protein phosphatases of type 1 and 2A. They are synthesised by the enzyme complex microcystin synthetase (MCS), which includes a non-ribosomal peptide synthetase (NRPS) and a polyketide synthase (PKS) (Nishizawa et al. 2000; Tillett et al. 2000). Both NRPS and PKS are made up of different modules, each one consisting of common domains required for addition of one peptide or ketide unit, and a variable number of other domains responsible for modification of the peptide/ketide backbone (Ansari et al. 2004). The Microcystis genes codifying for MCS, mcy , are organised in a cluster of two large bidirectional transcribed operons (mcyABC and mcyDEFGHIJ), spanning a region of about 55 kbp that include up to ten open reading frames (ORFs), as revealed by studies with two microcystin- producing Microcystis aeruginosa: PCC7806 (Tillett et al. 2000) and K139 (Nishizawa et al. 2000). The mcyABC genes encode five NRPS modules; the mcyD gene, two PKS modules, and the mcyE and mcyG genes, hybrid NRPS-PKS modules. The other genes (mcyF , -HIJ) are involved directly or indirectly in MCs synthesis, encoding a racemase (mcyF , Sielaff et al. 2003), a putative ABC transporter (mcyH, Pearson et al. 2004), a D-3 phosphoglycerate dehydrogenase (mcyI, Pearson et al. 2007), and an O-methyltransferase (mcyJ, Christiansen et al. 2003). The identification of toxic genotypes among non-toxic genotypes in environmental samples was long time impeded because they cannot be differentiated morphologically. Besides, the factors leading to cyanobacterial blooms with MC-producing (MC + ) or non MC-producing (MC - ) strains Y. Ouahid : F. F. del Campo (*) Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain e-mail: francisca.delcampo@uam.es Appl Microbiol Biotechnol (2009) 85:405–412 DOI 10.1007/s00253-009-2249-4