Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or real-time PCR.
The removal of target DNA by magnetic capture hybridization (MCH) from constituents inhibitory to amplification by polymerase chain reaction (PCR) was evaluated using Salmonella as the test pathogen. Hybrids were subjected to both conventional and quantitative real-time PCR (qPCR). When PCR inhibitors commonly found in water were added to the reaction, MCH-PCR increased the detection sensitivity on the order of 8 to 2,000-fold compared with the 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 qPCR reaction. The highest non-target DNA concentration interfered with the amplification by qPCR alone, while MCH-qPCR was unaffected. Average recovery of Salmonella DNA by MCH-qPCR was 31% using optimized 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.
Thompson
D E
DE
Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Rajal
V B
VB
De Batz
S
S
Wuertz
S
S
D008280:Magnetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D014481:United States; D014871:Water Microbiology; D014881:Water Supply / Q000032:analysis*