Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array

Abstract

The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.

FIG. 1

Summary of hybridization detected for the 23S rDNA PCR products of 95 pure cultures. The positions of the oligonucleotides are indicated in the lower right-hand strip. Their sequences are indicated in Table 2. The number of strains tested is indicated in the top portion of each strip. Filled circles represent strong hybridization, empty circles represent weak hybridization, and empty cells represent no hybridization detected.

FIG. 2

Results of the hybridization assay for 23S PCR amplifications from 12 blood culture bottles. Strip 0 was a DNA-negative PCR control. Strips 1 to 12 were PCR amplifications from bottles which subsequently grew bacteria identified as Proteus mirabilis (strip 1), CoNS (strips 2, 3, 7, 8, and 9), S. maltophilia (strip 4), E. coli (strip 5), P. aeruginosa (strip 6), S. aureus (strip 10), Corynebacterium spp. (strip 11), and E. faecium (strip 12). No hybridization is visible on strip 7 due to failure of the PCR. The oligonucleotides are listed in Table 2 and were arranged as shown in Fig. 1.