Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty

Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chai...

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Bibliographic Details
Published in:Analytical biochemistry 2007-02, Vol.361 (2), p.169-175
Main Authors: Kalogianni, Despina P., Goura, Sophia, Aletras, Alexios J., Christopoulos, Theodore K., Chanos, Michalis G., Christofidou, Myrto, Skoutelis, Athanasios, Ioannou, Penelope C., Panagiotopoulos, Elias
Format: Article
Language:English
Subjects:
23S rRNA
Arthroplasty
Bacteria - genetics
Bacteria - isolation & purification
Bacterial infection
Bacterial Infections - diagnosis
Cross Infection - diagnosis
Dipstick test
DNA Probes - chemistry
DNA, Bacterial - analysis
DNA, Ribosomal - analysis
Enterococcus
Escherichia coli
Gold - chemistry
Haemophilus
Humans
Joint Prosthesis - microbiology
Nanoparticles - chemistry
Polymerase Chain Reaction - methods
RNA, Ribosomal, 23S - genetics
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus pneumoniae
Synovial Fluid - microbiology
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Summary:Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2006.11.013