Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.
A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.
Jacobsen
C S
CS
Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Frederiksberg C, Copenhagen, Denmark.
D001431:Bacteriological Techniques; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D005798:Genes, Bacterial; D001467:Hordeum / Q000382:microbiology; D006812:Humic Substances / Q000302:isolation & purification; D008280:Magnetics; D008832:Microchemistry; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000235:genetics; D012988:Soil Microbiology
D015342:DNA Probes; D004269:DNA, Bacterial; D006812:Humic Substances