Interlaboratory comparison of Taq Nuclease Assays for the quantification of the toxic cyanobacteria Microcystis sp.
The application of quantitative real-time PCR has been proposed for the quantification of toxic genotypes of cyanobacteria. We have compared the Taq Nuclease Assay (TNA) in quantifying the toxic cyanobacteria Microcystis sp. via the intergenic spacer region of the phycocyanin operon (PC) and mcyB indicative of the production of the toxic heptapeptide microcystin between three research groups employing three instruments (ABI7300, GeneAmp5700, ABI7500). The estimates of mcyB genotypes were compared using (i) DNA of a mcyB containing strain and a non-mcyB containing strain supplied in different mixtures across a low range of variation (0-10% of mcyB) and across a high range of variation (20-100%), and (ii) DNA from field samples containing Microcystis sp. For all three instruments highly significant linear regression curves between the proportion of the mcyB containing strain and the percentage of mcyB genotypes both within the low range and within the high range of mcyB variation were obtained. The regression curves derived from the three instruments differed in slope and within the high range of mcyB variation mcyB proportions were either underestimated (0-50%) or overestimated (0-72%). For field samples cell numbers estimated via both TNAs as well as mcyB proportions showed significant linear relationships between the instruments. For all instruments a linear relationship between the cell numbers estimated as PC genotypes and the cell numbers estimated as mcyB genotypes was observed. The proportions of mcyB varied from 2 to 28% and did not differ between the instruments. It is concluded that the TNA is able to provide quantitative estimates on mcyB genotype numbers that are reproducible between research groups and is useful to follow variation in mcyB genotype proportion occurring within weeks to months.
Schober
Eva
E
Institute for Limnology, Austrian Academy of Sciences, 5310 Mondsee, Austria.
Werndl
Michael
M
Laakso
Kati
K
Korschineck
Irina
I
Sivonen
Kaarina
K
Kurmayer
Rainer
R
D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D005618:Fresh Water / Q000382:microbiology; D046931:Microcystis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010798:Phycocyanin / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000378:metabolism*; D014871:Water Microbiology
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D010798:Phycocyanin; D019914:Taq Polymerase; D003851:Deoxyribonucleases