Title
Real-time PCR monitoring of toxic cyanobacteria in Bulgarian water samples
Poster
Description
Viktoria Ilieva 1 , Tzveta Georgieva 2 , Magdalena Kondeva-Burdina1
1Faculty of Pharmacy, Medical University of Sofia 2National Centre of Public Health and Analises
1Faculty of Pharmacy, Medical University of Sofia 2National Centre of Public Health and Analises
Subject
Some of Cyanobacteria (Microcystis, Anabena, Planktothrix) can produce toxins in
concentrations harmful humans and animals. Some of the most dangerous cyanotoxins
are mycrocystins. As a risk factor, early detection is required at low concentrations. The
applications at low levels. The World Health Organization recommends the maximum
acceptable value for drinking watersto be up to 1 μg / l and 20 μg / l for bathing water for
one of the most dangerous cyanotoxins – microcystines. Chronic exposure to cyanotoxins,
for example – microcystines, is considered to be a risk factor for the development of
muscle paralysis, neurodegenerative diseases and liver diseases. In Bulgaria the first
exploration of cyanotoxins is dated back to 2004, and it includes 15 water basins. The
National Center for Public Health and Analysis in Bulgaria controls and examines these
toxins. According to Annex II of the Framework Directive 2000/60 / EU, amended by
Regulation No. 13/2007 on surface water and assessment are absolutely necessary, but at
this stage there are no tolerable limits for cyanotoxins. Methods for the investigation and
monitoring of cyanobacteria and their toxins are under development. Because of their
toxicity to human health, they need to be early detected in low concentrations with a
precise and highly sensitive method. Therefore, the presentstudy focuses on the isolation
of sufficient DNA with good quality. This is a necessary condition for Polymerase Chain
Reaction in real-time. The goal of the academic research is to introduce appropriate
molecular genetic methods for isolating DNA from aqueous samples and early detection
of the cyanoxins by in-real-time PCR. The aim is to cover more Bulgarian water basins
during the survey. The isolation of sufficient DNA with good quality is a necessary
condition for obtaining real result in subsequent RealTime PCR. The presence of inhibitors
in the extracted DNA samples may result in false positive or negative results. This in vitro
genetic method is chosen because of its high accuracy and low limit of detection. Samples
of "Bistritsa" , “Pchelin” and "Studena" dams have been studied. For the control samples
and biological samples were used material from “Durankulak” and “Shabla” dams , where
was proven presence of toxic cyanobacteria by microscopic observation and
chromatographic analysis. The bacterial DNA is isolated by two methods: 1. a xanthogenic
method optimized for the purposes of this thesis and 2. a commercial kit. A comparative
analysis of the two sampling methods was made. Real-time polymerase chain reaction
(real-time PCR) analysis was performed using the TaqMan and SYBRGreen approach. To
assess the presence of bacterial DNA, specific primers for the detection of Cyanobactria
were selected. Genetic markers for identifying the toxic species Microcystis aeruginosa
are selected portions of the microcysty genetic cluster mcy: mcyA, mcyB, cya359. It is
planned for the next two years the mechanism of neurotoxicity and hepatotoxicity to be
explored in details. The researches will be made in the scientific laboratories at the Faculty
of Pharmacy at the Medical University - Sofia.
concentrations harmful humans and animals. Some of the most dangerous cyanotoxins
are mycrocystins. As a risk factor, early detection is required at low concentrations. The
applications at low levels. The World Health Organization recommends the maximum
acceptable value for drinking watersto be up to 1 μg / l and 20 μg / l for bathing water for
one of the most dangerous cyanotoxins – microcystines. Chronic exposure to cyanotoxins,
for example – microcystines, is considered to be a risk factor for the development of
muscle paralysis, neurodegenerative diseases and liver diseases. In Bulgaria the first
exploration of cyanotoxins is dated back to 2004, and it includes 15 water basins. The
National Center for Public Health and Analysis in Bulgaria controls and examines these
toxins. According to Annex II of the Framework Directive 2000/60 / EU, amended by
Regulation No. 13/2007 on surface water and assessment are absolutely necessary, but at
this stage there are no tolerable limits for cyanotoxins. Methods for the investigation and
monitoring of cyanobacteria and their toxins are under development. Because of their
toxicity to human health, they need to be early detected in low concentrations with a
precise and highly sensitive method. Therefore, the presentstudy focuses on the isolation
of sufficient DNA with good quality. This is a necessary condition for Polymerase Chain
Reaction in real-time. The goal of the academic research is to introduce appropriate
molecular genetic methods for isolating DNA from aqueous samples and early detection
of the cyanoxins by in-real-time PCR. The aim is to cover more Bulgarian water basins
during the survey. The isolation of sufficient DNA with good quality is a necessary
condition for obtaining real result in subsequent RealTime PCR. The presence of inhibitors
in the extracted DNA samples may result in false positive or negative results. This in vitro
genetic method is chosen because of its high accuracy and low limit of detection. Samples
of "Bistritsa" , “Pchelin” and "Studena" dams have been studied. For the control samples
and biological samples were used material from “Durankulak” and “Shabla” dams , where
was proven presence of toxic cyanobacteria by microscopic observation and
chromatographic analysis. The bacterial DNA is isolated by two methods: 1. a xanthogenic
method optimized for the purposes of this thesis and 2. a commercial kit. A comparative
analysis of the two sampling methods was made. Real-time polymerase chain reaction
(real-time PCR) analysis was performed using the TaqMan and SYBRGreen approach. To
assess the presence of bacterial DNA, specific primers for the detection of Cyanobactria
were selected. Genetic markers for identifying the toxic species Microcystis aeruginosa
are selected portions of the microcysty genetic cluster mcy: mcyA, mcyB, cya359. It is
planned for the next two years the mechanism of neurotoxicity and hepatotoxicity to be
explored in details. The researches will be made in the scientific laboratories at the Faculty
of Pharmacy at the Medical University - Sofia.