Clinically applicable multiplex PCR for four middle ear pathogens.

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.