Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children

• Published in: Journal of microbiological methods 2007, Vol.68 (1), p.88-93
• Main Authors: Rosey, Anne-Laure, Abachin, Eric, Quesnes, Gilles, Cadilhac, Céline, Pejin, Zagorka, Glorion, Christophe, Berche, Patrick, Ferroni, Agnès
• Format: Article
• Language: English
• Subjects: 16S rDNA PCR; Adolescent; Arthritis, Infectious - diagnosis; Arthritis, Infectious - microbiology; Articular fluids; Bacterial arthritis and osteitis; Bacterial diseases; Biological and medical sciences; Child; Child, Preschool; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Fluorescent Dyes - chemistry; Human bacterial diseases; Humans; Infant; Infectious diseases; Kingella kingae; Medical sciences; Organic Chemicals - chemistry; Polymerase Chain Reaction - methods; Salmonella; Sequence Analysis, DNA; Staphylococcus aureus; Streptococcus pneumoniae; Streptococcus pyogenes; SYBR Green; Synovial Fluid - microbiology
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2006.06.010

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria ( Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.