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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.

Identification of pathogenic Mycobacterium species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two M. avium-intracellulare complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of M. tuberculosis , MAC, M. fortuitum , M. chelonae , M. abscessus , M. kansasii , M. gordonae , M. scrofulaceum , M. szulgai , M. vaccae , M. xenopi , M. terrae , M. flavescens , M. smegmatis , M. malmoense , M. simiae , M. marinum , M. ulcerans , M. gastri , and M. leprae . All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus Mycobacterium . Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for M. marinum and M. ulcerans , which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.

An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB , katG , and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One-hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates.