Clinically applicable multiplex PCR for four middle ear pathogens

The multiplex PCR method for the detection of Alloiococcus otitidis, Haemop hilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (P. H . Hendolin, A. Markkanen, J. Mikoski, and J. J. Wahlfors, J. Clin. Microbio l. 35:2854-2858, 1997) in middle ear effusions (MEEs) was modified to be be tter suited for clinical use. To detect false-negative results, an internal amplification was added to the reaction, and to prevent carryover contamin ation, the dUTP-uracil-N-glycosidase system was incorporated into the proce dure. Labor was minimized by using the heat-activatable AmpliTaq Gold polym erase in order to circumvent manual hot start and by detecting the amplific ation products on an automated sequencer. The performance of the improved p rotocol was verified with MEEs from patients with otitis media with effusio n. In addition, a Ligase detection reaction (LDR) was developed for confirm ation of the PCR products. The modifications increased the reliability of t he protocol and the hands-off time significantly. However, when two DNA ext raction 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 sulfa te-NaOH-chaotropic salt (83 versus 27%; P < 0.001). The LDR was found to be 100% specific. In ail, the results demonstrate the feasibility of the rapi d (7-h) multiplex PCR method for routine laboratory use.