Application of TaqMan probes in end-point fluorimetry for detection of pathogenic bacteria by polymerase chain reaction
Abstract number: 902_p704
5-Nuclease polymerase chain reaction in a conventional thermal cycler and a subsequent fluorescence measurement in a 96 well fluorimeter were developed and successfully carried out in flat-bottom microtubes.
Complete reaction system was simply transferred from real-time PCR to new instrumental conditions. Specific primers and specific TaqMan probes (fluorescent dye 6-FAM, quenching dye TAMRA) for Salmonella sp. and E. coli strains were used. When finishing the PCR, the fluorescence was measured in endpoint mode in fluorescence reader equipped with an excitation filter with a pass maximum of 492 nm and an emission filter with a pass maximum of 520 nm from the bottom orientation. To define the positivity threshold, three negative control samples (containing no DNA template) were used. Mean value and the standard deviation (SD) were calculated and the positivity threshold was set to (mean + 2 SD).
In these conditions, consistent results were obtained when PCR was done with purified Salmonella Enteritidis and E. coli DNA or with culture lysates. When lysates of series of decimally diluted cultures were analysed a detection limit of 10exp4 CFU/mL was determined, the same sensitivity as with real-time PCR and the same or one order of magnitude higher than with the gel electrophoresis were determined.
The method proved to be a fast and contamination-free alternative to gel electrophoresis and an inexpensive alternative to real-time PCR."
|Session name:||XXIst ISTH Congress|
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