A gyrB oligonucleotide microarray for the specific detection of pathogenic Legionella and three Legionella pneumophila subsp.

Abstract

Among the 50 species and 70 serogroups of Legionella identified, Legionella pneumophila, comprising three subsp. (subsp. pneumophila, subsp. fraseri, and subsp. pasculleii), is recognized as the major cause of epidemic legionellosis. Rapid and reliable assays to identify pathogenic Legionella spp., and the three L. pneumophila subsp. in particular, are in great demand. In this study, we analyzed the gyrB genes of eleven Legionella spp. and subsp., comprising L. anisa, L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. longbeachae, L. micdadei, L. waltersii, L. pneumophila subsp. pneumophila, L. pneumophila subsp. fraseri, and L. pneumophila subsp. pasculleii. We developed a rapid oligonucleotide microarray detection technique to identify accurately these common pathogenic Legionella spp. and L. pneumophila subsp. To detect multiple Legionella species with high specificity, 31 reproducible probes were designed in the array. Sixty-one strains were analyzed in total, including 37 target pathogens and 24 non-target bacterial species used to validate the microarray. The sensitivity of the detection was 1.0ng using genomic DNA of three Legionella spp., L. anisa, L. dumoffii, and L. waltersii, or 13CFU/100mL using the cultured L. pneumophila subsp. pneumophila. Eight isolated strains were tested using the microarray with 100% accuracy. The data indicated that the technique is an efficient method to diagnose and detect Legionella spp. and subsp. in basic microbiology, clinical diagnosis, epidemiological surveillance, and food safety applications. In addition, a phylogenetic study based on the gyrB gene revealed the genetic relationship among the different Legionella spp. and subsp.