Magnetic capture hybridisation for improved PCR detection of Nectria galligena from lignified apple extracts

In order to reduce the effects of inhibitors present in DNA extracts from lignified apple tissues, a magnetic capture-hybridisation PCR (MCH-PCR) technique was developed for Nectria galligena using the ITS 1 region of the rRNA gene repeats as target. The trapping reagent used to coat the magnetic beads was an 81 bp single-stranded DNA oligonucleotide biotin-labelled on the 5'-terminal and designed to be complementary to part of the rRNA gene ITS 1 region of N. galligena. For specificity, the probe was located from 14 bp downstream from the 3'-terminal nucleotide of the N. galligena forward primer Chi to the last ITS 1 nucleotide immediately upstream of the 5.8S rRNA gene. Following hybridisation in a total DNA extract of woody tissue, magnetic recovery of the bead-oligomer-template conjugate separated target template from other DNA species and inhibitory compounds. Magnetic capture-hybridisation was followed by PCR amplification with the previously designed species-specific primers, Ch1 and Ch2, Application of the MCH-PCR technique resulted in increased levels of sensitivity and reliability when compared to PCR without MCH when used on total DNA extracts from lignified tissues.