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Clinically applicable multiplex PCR for four middle ear pathogens
P H Hendolin1, L Paulin, J Ylikoski
1Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. Panu.Hendolin@Helsinki.fi
Abstract:
The multiplex PCR method for the detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (P. H. Hendolin, A. Markkanen, J. Ylikoski, and J. J. Wahlfors, J. Clin. Microbiol. 35:2854-2858, 1997) in middle ear effusions (MEEs) was modified to be better suited for clinical use. To detect false-negative results, an internal amplification was added to the reaction, and to prevent carryover contamination, the dUTP-uracil-N-glycosidase system was incorporated into the procedure. Labor was minimized by using the heat-activatable AmpliTaq Gold polymerase in order to circumvent manual hot start and by detecting the amplification products on an automated sequencer. The performance of the improved protocol was verified with MEEs from patients with otitis media with effusion. In addition, a ligase detection reaction (LDR) was developed for confirmation of the PCR products. The modifications increased the reliability of the protocol and the hands-off time significantly. However, when two DNA extraction protocols were compared, gram-negative bacteria were detected more often in phenol-treated MEEs (94 versus 46%; P < 0.001), and gram-positive bacteria were detected more often in MEEs dissolved in sodium dodecyl sulfate-NaOH-chaotropic salt (83 versus 27%; P < 0.001). The LDR was found to be 100% specific. In all, the results demonstrate the feasibility of the rapid (7-h) multiplex PCR method for routine laboratory use.
Insights
This study optimized a multiplex PCR method for detecting common middle ear infection bacteria in middle ear effusions (MEEs). The enhanced protocol improves reliability and reduces hands-on time for clinical laboratory use.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
Background:
- The multiplex PCR method for detecting bacteria in middle ear effusions (MEEs) required optimization for clinical settings.
- Accurate and rapid detection of pathogens in MEEs is crucial for diagnosing otitis media.
Purpose of the Study:
- To modify and enhance a multiplex PCR protocol for improved clinical utility in detecting bacterial pathogens in MEEs.
- To increase the reliability and efficiency of the multiplex PCR assay for routine laboratory diagnostics.
Main Methods:
- Incorporated an internal amplification control to prevent false-negative results and a dUTP-uracil-N-glycosidase system to prevent contamination.
- Utilized heat-activatable AmpliTaq Gold polymerase and automated sequencing to minimize labor and manual steps.
- Developed a ligase detection reaction (LDR) for PCR product confirmation and compared two DNA extraction protocols.
Main Results:
- The improved multiplex PCR protocol demonstrated increased reliability and significantly reduced hands-off time.
- DNA extraction method significantly impacted bacterial detection rates: phenol-treated MEEs favored gram-negative bacteria (94%), while sodium dodecyl sulfate-NaOH-chaotropic salt favored gram-positive bacteria (83%).
- The ligase detection reaction (LDR) achieved 100% specificity, confirming PCR product accuracy.
Conclusions:
- The modified 7-hour multiplex PCR method is feasible for routine clinical laboratory use.
- The enhancements address key challenges in molecular diagnostics, including reliability and contamination prevention.
- The choice of DNA extraction protocol is critical for optimizing the detection of different bacterial types in MEEs.