ABSTRACT
Ralstonia
solanacearum is the causative agent of bacterial wilt in many
important crops. A specific and sensitive PCR detection method that
uses primers targeting the gene coding for the flagella subunit,
fliC, was established. Based on the first fliC gene
sequence of R. solanacearum strain K60 available at GenBank,
the Ral_fliC PCR primer system was designed; this
system yielded a single 724-bp product with the DNAs of all of the
R. solanacearum strains tested. However, R. pickettii
and four environmental Ralstonia isolates also yielded
amplicons. The Ral_fliC PCR products obtained with 12
strains (R. solanacearum, R. pickettii, and
environmental isolates) were sequenced. By sequence alignment,
Rsol_fliC primers specific for R.
solanacearum were designed. With this primer system, a specific
400-bp PCR product was obtained from all 82 strains of R.
solanacearum tested. Six strains of R. pickettii and
several closely related environmental isolates yielded no PCR product;
however, a product was obtained with one Pseudomonas syzygii
strain. A GC-clamped 400-bp fliC product could be
separated in denaturing gradient gels and allowed us to distinguish
P. syzygii from R. solanacearum. The
Rsol_fliC PCR system was applied to detect R.
solanacearum in soil. PCR amplification, followed by Southern blot
hybridization, allowed us to detect about one target DNA molecule per
PCR, which is equivalent to 103 CFU g of bulk
soil−1. The system was applied to survey soils from
different geographic origins for the presence of R.
solanacearum.
4.2.2 InCheck System: a Highly Integrated Silicon Labonchip for Sample Preparation, PCR Amplification and Microarray Detection Towards the Molecular Diagnostics Pointofcare