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The Effects of Pretreatments on qPCR Detection of Bacteria - Bacteria Indicator, Enterococcus as a Model
Shin Giek Goh and Karina Yew-Hoong Gin
Current recreational water guidelines (e.g. USEPA and WHO) recommend enterococci as the indicator for both freshwaters and marine waters. Culture-based method has been used for decades to quantify the presence of enterococci. In year 2012, USEPA guideline for recreational water included qPCR as the alternative method to detect enterococci. qPCR method does not require long incubation time as of culture-based method. In addition, it offers the advantages of high specificity and sensitivity detection. Nevertheless, qPCR hampered by three main limiting factors. There are limited target cell in small volume of sample being analyzed by qPCR, the presence of inhibitors that can easily affect the sensitive detection of qPCR and the persistence of free DNA (DNA released from dead cell) that caused the false-positive signal in qPCR. As such, the upstream treatments are crucial to the accuracy of qPCR detection. Three different pre-concentration methods (i) filtration with nylon membrane; (ii) filtration with polycarbonate membrane and (ii) centrifugation, were examined for their recovery rate. Filtration with polycarbonate membrane was found to give higher recovery efficiency and consistence results regardless of sample matrices. Conventional DNA extraction and two commercially available DNA extraction kits were evaluated according to the purity of extracted DNA and the relative recovery efficiency of extraction. Results show that the commercial kit, QIAamp® DNA Mini Kit (Qiagen), gave the best performance. The application of silica membranes in QIAamp® DNA Mini Kit was proven to enhance the DNA recovery with minimum interference of inhibitors. Ethidium monoazide (EMA) and propidium monoazide (PMA) were evaluated for their performance in reducing the false-positive signal in qPCR. PMA was appear to provide better option to reduce the false-positive detection of DNA in a membrane compromised cell.