Bourne, D.G., Blakeley, R.L., Riddles, P., and Jones, G.J. (2005) Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR. Marine and Freshwater Research, 56 (8). pp. 1127-1135.

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Abstract

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation ( FISH) techniques were developed for the detection of a Sphingomonas bacterium ( strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA ( rDNA) of strain MJ-PV. A 16S ribosomal RNA ( rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

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