The rtxA toxin gene of Kingella kingae: a pertinent target for molecular diagnosis of osteoarticular infections

Abstract

Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection.

Fig. 1.

Melting curve analyses of the PCR products obtained from the amplification of the rtxA gene of Kingella kingae with the ABI PRISM 7000 (Applied Biosystems) (A), the SmartCycler (Cepheid) (B), and the LightCycler (Roche Diagnostics) (C). The temperature (°C) is indicated on the x axis, and the derivative of the fluorescence is indicated on the y axis. The peaks indicate the melting points of the respective amplicons.