Fast and sensitive method for the detection of bacteria in synovial fluid by multiplex PCR

Background

Bacterial infections are common and are known to be involved in many diseases including arthritis. While some methods for detecting bacteria are time-consuming, others are faster but lack specificity and/or sensitivity.

Methods and results

We employed a new method for DNA isolation and compared the results with those of an established method. We show that an equivalent of fewer than 10 bacteria of Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus, Staphylococcus epidermitis (SE) or bacteria in general can be detected in less than 2 hours.

We found that lyses in a SDS/β-mercaptoethanol buffer and exposure to microwaves is as well suited for detecting fewer than 10 microor-ganisms per sample as a longer, more elaborate and more expensive method using specific enzymes. We further confirmed the sensitivity of our multiplex PCR method by recovery experiments using known amounts of bacteria. Such experiments showed that bacteria present in single-digit quantities can be detected even in viscous solutions such as synovial fluid. We further showed that the chosen primer pairs are suited for multiplex PCR, the simultaneous testing of samples for the presence of SA, SE, and/or bacteria in general. We also showed that the assay conditions are suitable for detecting the presence of methicillin-resistant forms of SA. The specificity of the PCR was confirmed by comparison of the actual size with the calculated length of the fragment, and by sequencing of the PCR product.

Conclusion

Taken together, our method of rapid DNA isolation and the optimized PCR program make it possible to confirm the presence or absence of minute amounts of bacteria. Employing real-time PCR would shorten this procedure even further. This method might therefore contribute to more timely and specific interventions.