The development and application of qPCR-based system to diagnose the presence of harmful cyanobacteria and their metabolites in drinking water sources

摘要

Lakes and reservoirs represent important drinking water sources in Taiwan and many other countries Due to improper management of nutrients in their watersheds many reservoirs are facing the risk of eutrophication increasing the possibility of cyanobacterial blooms in the source waters Some of them may produce cyanotoxins and odorants as secondary metabolites such as microcystins (MCs) cylindrospermopsin (CYN) and 2-methylisoborneol (2-MIB) Drinking water contaminated with cyanotoxins and odorants may affect public health and consumers’ perception to public water supply Therefore it is very important to understand the presence of cyanobacteria and their metabolites in water supplies In addition to correlate the presence of a toxin/odorant in a given reservoir to a specific cyanobacterial species isolation of strains from each environmental sample was a necessary step The isolated strains can then be easily screened for the production of toxins/odorants and be studied morphologically and genetically in order to assign them to a particular species However loss of general characteristic such as morphology physiology biochemistry and genetic properties during serial sub-culturing has been evidenced in several cyanobacterial cultures Therefore it is important to have a reliable and simple technique for long-term preservation after acquiring or isolating cyanobacterial culture Currently when water utilities are faced with cyanobacteria problems in the source water to confirm the risk associated with cyanotoxins and odorants the standard procedures include sample collection and delivery laboratory analysis and reporting The whole procedures usually take more than 24 hours In this case the contaminated water may have entered into public water supplies Therefore a rapid method to quickly provide the potential risk of cyanobacteria associated toxins and odorants would be very usefully for water utilities to justify the quality of their drinking water This study aimed to establish a quantitative real-time polymerase chain reaction (qPCR) to quickly determine the risk of cyanobacteria associated toxins (MCs and CYN) and odorants (2-MIB) in fresh water bodies based on the detection of cyanotoxin and odorant-producing cyanobacterial genes Concurrently an enzyme-linked immunosorbent assay (ELISA) and a solid-phase microextraction (SPME) concentration followed by a gas chromatograph-mass selective detector (GC-MSD) was employed to measure cyanotoxins and odorants produced by cyanobacteria respectively Samples were also enumerated for cyanobacteria cells with microscope In addition the efficacy of the various cryoprotective agents was compared for several isolates of cyanobacteria This study highlighted that the 5 % dimethyl-sulphoxide (DMSO) for cryopreservation was shown to be a preferable choice of cryopreservative for most strains tested This qPCR based approaches have been successfully applied in 29 drinking-water sources in Taiwan and her offshore islands Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R2 = 0 392?0 740) There are also statistically significant correlations between mibC genes and 2-MIB concentrations (range of coefficients of determination R2 = 0 473?0 479) The results demonstrate that the developed qPCR based approaches were able to quantify the targeted cyanobacteria and toxin/odorant producing genes In particular the obtained gene information correlated well with cell numbers enumerated with microscopy toxin concentrations measured with ELISA and 2-MIB concentration measured with GC/MS This scheme should provide timely information to managers of reservoirs and water utilities to diagnose the potential risk associated with toxigenic cyanobacteria cyanotoxins and 2-MIB in drinking water lakes and reservoirs

獎項日期	2017 8月 7
原文	English
監督員	Tsair-Fuh Lin (Supervisor)