Using oligonucleotide suspension arrays for laboratory identification of bacteria responsible for bacteremia

Abstract

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.

Fig. 1

A histogram of representative hybridization signals generated by hybridization of bead-immobilized probes to PCR products from all bacteria studied AH: Aeromonas hydrophila; BC: Burkholderia cepacia; EF1: Enterococcus faecalis; EF2: Enterococcus faecium; HI: Haemophilus influenzae: KO: Klebsiella oxytoca; LM: Listeria monocytogenes; PA: Pseudomonas aeruginosa; PM: Proteus mirabilis; ST: Salmonella typhimurium; SM: Stenotrophomonas maltophilia; SA: Staphylococcus aureus; SE: Staphylococcus epidermidis; SH: Staphylococcus haemolyticus; SP: Streptococcus pneumoniae

Fig. 2

The detection limit of the suspension array with 15 bead sets for Listeria monocytogenes NICPBP54001. PCR product of 9.3×10⁷ CFU/ml at 26 cycles and amplicons at 35 thermal cycles from different concentrations (9.3×10⁷, 9.3×10⁶ and 9.3×10⁵ CFU/ml) and template-negative control (PCR NC), were hybridized to the multiplex array and analyzed by LiquiChip reader. On the basis of 9c positive signals, Listeria monocytogenes was identified. The detection limit at 35 cycles for Listeria monocytogenes was 9.3×10⁶ CFU/ml C ₁: 9.3×10⁷ (26 cycles); C ₂: 9.3×10⁷ (35 cycles); C ₃: 9.3×10⁶ (35 cycles); C ₄: 9.3×10⁵ (35 cycles); C ₅: PCR NC (35 cycles)