Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or real-time PCR

D.E. THOMPSON; V.B. RAJAL; S. DE BATZ; S. WUERTZ

Journal of Water and Health

IWA Publishing

Año: 2006 vol. 4 p. 67 - 75

1477-8920

Detection of pathogens in environmental water samples requires specific and sensitive methods. Magnetic capture hybridization (MCH) separates specific target DNA from other DNA and interfering compounds. In this study, inhibitor removal using biotin-labelled oligonucleotide probes and streptavidin coated magnetic beads was evaluated using Salmonella as the test pathogen. Hybrids were subjected to nucleic acid amplification, using both conventional and quantitative real-time (TaqMan) PCR. PCR inhibitors commonly found in environmental water were added in varying amounts to a fixed concentration of Salmonella DNA.  MCH-PCR increased the detection sensitivity on the order of 8 to 2,000-fold compared to the reaction system using only PCR. To determine the selectivity of MCH for target DNA (Salmonella), different amounts of non-target DNA (Escherichia coli) were added to the TaqMan reaction mixture. The highest non-target DNA concentration using only TaqMan interfered with the amplification, while MCH-TaqMan was unaffected. A method based on the combination of MCH and quantitative real-time PCR (qPCR) was developed and evaluated. Recovery of Salmonella DNA ranged from 2-50% depending on the buffers, washing solutions, and enzymatic digestion. A recovery function was proposed in order to calculate the real cell number based on the measured value. Preliminary testing confirmed the suitability of this method for analysis of natural waters.