CYANOBACTERIAL TOXINS - Cyanobacteria - page 551

MEKC has proved benefi cial as it can overcome the problems of interference by high protein content
of the samples during CZE separation. MEKC proved more advantageous for the analysis of CYN
because of the retention of proteinaceous material by anionic micelles. Thus CZE and MEKC are
useful, rapid and sensitive physicochemical assays for simultaneous determination of MC-LR, CYN
and anatoxin-a (Vaas et al ., 2004).
Analysis of STXs, produced by A . circinalis and other cyanobacteria, in the range of 10-70 ppb
was demonstrated by a single run HPLC method by employing ion exchange as a separation mode
with a novel mobile phase system coupled to post column oxidation and fl uorescence detection.
During the analysis of STXs either the presence of cyanobacterial matrix or natural water constituents
did not interfere in any manner (Papageorgiou et al ., 2005).
3) Molecular Probes: A number of molecular probes have been identifi ed during recent years to
identify the toxic blooms both from laboratory cultures and natural samples. The utility of such
molecular probes is unequivocal in the identifi cation of a particular toxic cyanobacterial species but
these require trained personnel and also involve lot of expenditure with reference to sophisticated
instruments and very costly chemicals that an ordinary public health institution can afford.
Nevertheless these techniques are greatly helpful in the identifi cation of toxic cyanobacteria up to
species level. These are discussed here.
i) PC-IGS sequences : The utility of intergeneric spacer (IGS) between the bilin subunit genes, i.e.
β ( cpcB ) and α ( cpcA ) of the phycocyanin (PC) operon has been examined for the identifi cation of
cyanobacteria to the strain level. Thus PC-IGS sequence was amplifi ed by PCR from DNA extracted
from the environmental samples. Restriction endonuclease digestion of the PCR amplifi ed products
yielded many DNA profi les specifi c to different cyanobacteria. As for example, restriction enzyme
profi les for MspI , RsaI and Taq1 could be assigned to eight genera (40 strains). Genus Microcystis
could be identifi ed with restriction enzymes AluI , CfoI and HaeIII whereas Anabaena was identifi ed
with digestion profi les provided by AluI , CfoI and ScrFI (Neilan et al ., 1995).
ii) RAPD and PCR of 10-mer sequences : Random oligonucleotide primers of 10 nucleotides long
combined with PCR have given a molecular tool that is useful for recognition of DNA from various
living organisms. The randomly amplifi ed polymorphic DNA (RAPD) technique in combination
with PCR helped in identifying the DNA profi les specifi c to the toxic cyanobacteria tested to the
level of genus, species and strain. The genera Anabaena and Microcystis could be easily distinguished
(Neilan, 1995).
iii) Heptamer repeats : The genomes of fi lamentous, heterocystous cyanobacteria possess short
tandemly repeated repititive (STRR) DNA sequences between or within genes. The genome of
Anabaena sp. strain PCC 7120 consists of such heptamer repeats at many sites, i.e. in and around
PC operon (Belknap and Haselkorn, 1987), after the atpBE operon (Curtis, 1987), after the psbB gene
(Lang and Haselkorn, 1989), within the nifB operon (Mulligan and Haselkorn, 1989) and within nifJ
gene (Bauer et al ., 1993). The heptamer repeat sequence CCCCAGT found in Anabaena sp. strain
PCC 7120 is common with Anabaena and Nostoc strains. Genomes of non-heterocystous Oscillatoria
sp. and Microcystis sp. are devoid of such heptamer sequences. It is concluded that this sequence
is mobile between Anabaena and Nostoc strains (Bauer et al ., 1993). Rouhiainen et al . (1995) used
RFLP analysis using STRR to distinguish Anabaena isolates producing hepatotoxins from those of
neurotoxin-producing strains and from Nostoc spp.