Development and Evaluation of Oligonucleotide Chip Based on the 16S-23S rRNA Gene Spacer Region for Detection of Pathogenic Microorganisms Associated with Sepsis

Abstract

Oligonucleotide chips targeting the bacterial internal transcribed spacer region (ITS) of the 16S-23S rRNA gene, which contains genus-and species-specific regions, were developed and evaluated. Forty-three sequences were designed consisting of 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30 species-specific probes. The specificity of the probes was confirmed using bacterial type strains including 54 of 52 species belonging to 18 genera. The performance of the probes was evaluated using 825 consecutive samples that were positive by blood culture in broth medium. Among the 825 clinical specimens, 708 (85.8%) were identified correctly by the oligonucleotide chip. Most (536 isolates, or 75.7%) were identified as staphylococci, Escherichia coli, or Klebsiella pneumoniae. Thirty-seven isolates (4.5%) did not bind to the corresponding specific probes. Most of these also were staphylococci, E. coli, or K. pneumoniae and accounted for 6.3% of total number of the species. Sixty-two specimens (7.5%) did not bind the genus-or species-specific probes because of lack of corresponding specific probes. Among them, Acinetobacter baumannii was the single most frequent isolate (26/62). The oligonucleotide chip was highly specific and sensitive in detecting the causative agents of bacteremia directly from positive blood cultures.

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

“…Various target regions are used to identify pathogens in oligonucleotide microarray methods. These regions include 16S rDNA, 23S rDNA and 16S-23S rDNA internal transcribed spacer region (ITS) (14,16,26,27). The 23S rDNA genes appear to be the best performing regions as target sequences.…”

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

“…Mori et al (2004) suggested that phylogenetic analysis based on tripeptidase (PepT) and dipeptidase (PepV) genes may aid in a more precise index of classification of L. lactis subspecies. Beside 16S rRNA gene, 16S-23S rRNA gene intergenic spacer region or 23S rRNA gene have also been used to identify bacterial species (Blaiotta et al 2002;Mohania et al 2008;Kim et al 2010). However, there are no satisfactory databases for these genes for the development of L. lactis identification methods, except for 16S rRNA gene.…”

DNA Microarrays for Pathogen Detection

“…A DNA microarray for the detection 30 bacteria associated with sepsis contained 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30 species-specific probes directed against the 16S-23S rRNA ITS region. The microarray was validated with 825 positive blood culture samples from which 708 (85.8%) were identified correctly by the oligonucleotide chip [27]. Negoro et al [30] developed a DNA microarray which covered 72 bacteria which are often identified in positive blood cultures in combination with a PCR using universal PCR primers to amplify a variable region of the 16S rDNA.…”