Domingues, Lucília

Lima, Lígia

Mendes, D.

Teixeira, J. A.

Universidade do Minho: RepositoriUM

Please return your abstract to Maggi Churchouse, Conference Secretariat in the first instance, maggi@FIPS2011.org Development of molecular and enzymatic methods for the detection of total coliforms and Escherichia coli in water samples  Diana Mendesa, Lígia Limab, José A. Teixeiraa, Lucília Dominguesa a IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal b Frilabo, Rua S. Roque da Lameira, 71 - 4350-307 Porto Portugal   Session: C   Outbreaks of waterborne diseases remain a major challenge to public health providers, claiming millions of lives annually, worldwide. The need for more rapid, sensitive and specific tests is essential; not only for water industry, but for a better public safety.  Therefore, the aim of this research is to design commercial kits (enzymatic and molecular), at request and in collaboration with a Portuguese company, with the ability to detect E. coli and total coliforms in water samples. In order to be competitive in the market, these kits seek distinction for being fast to obtain the results, of simple handling, with low cost, high efficiency and sensitivity (1 CFU). In all kits to be developed, it has been explored both the qualitative and quantitative possibilities of detection. The most used in the microbiological water quality monitoring, nowadays, are the enzymatic method-based kits. These have commercial advantage over conventional methods (Multiple-Tube Fermentation, Membrane Filtration) due to short time response (18h to 24h), very simple handling and of relative low cost. Therefore, the kits being developed are based on the detection of the enzymes β-galactosidase, which catalyses the hydrolysis of a chromogenic substrate into a yellow coloured product indicating the presence of total coliforms; and β-glucuronidase (specific for E. coli), which catalysis the hydrolysis of a fluorogenic substrate into a blue fluorescent product, indicating the presence of faecal coliforms and, consequently, the possible presence of pathogenic organisms. The results are now visible in 18 h for 1 CFU.  PCR has distinct advantages over culture and other standard methods for the detection of indicators of microbiological pathogens: higher level of sensitivity and specificity without the need for a complex cultivation step or additional confirmation, rapidity, accuracy, capacity to detect small amounts of target nucleic acid in a sample and the ability to detect the bacteria known as viable but non-culturable. For this type of application, nucleic acid extraction is one of the most critical factors since cultivation is passed up in order to reduce the assay time and, as a consequence, obtaining a good quality nucleic acid template free of any inhibitory substances becomes challenging. In this work, it was intended to develop an efficient protocol for nucleic acid extraction and evaluate the effectiveness of PCR as a molecular method for the detection of total coliforms and E. coli, as well as its possible adaptation to microbiological water quality monitoring. Primers based on the lacZ, lamB and uidA genes were used and the pre-detection cell-culture step was avoided so that faster results could be achieved. At this moment, we have accomplished a sensitivity level of 676 CFU in 8 h.  PCR technology offers now a new option known as Real-Time PCR, which has been the most explored among PCR techniques in the last few years due to the capability of a much more rapid detection and enumeration of the target microorganism, in real-time, with no need for electrophoresis or additional time to detect the PCR products, as well as no confirmatory steps are needed. A new protocol is being developed in order to improve the detection of these indicator microorganisms, with the perspective to transform the microbiological water quality monitoring. 

Development of molecular and enzymatic methods for the detection of total coliforms and Escherichia coli in water samples

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Universidade do Minho: RepositoriUM