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Journal of Clinical Microbiology, January 2000, p. 125-132, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Clinically Applicable Multiplex PCR for Four Middle Ear Pathogens

Panu H. Hendolin,1,* Lars Paulin,1 and Jukka Ylikoski2

Institute of Biotechnology, University of Helsinki, 00014 Helsinki,1 and Department of Otorhinolaryngology, Helsinki University Central Hospital, 00290 Helsinki,2 Finland

Received 26 July 1999/Returned for modification 9 September 1999/Accepted 22 September 1999

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.

* Corresponding author. Mailing address: Institute of Biotechnology, University of Helsinki, P.O. Box 56, Viikinkaari 9, 00014 Helsinki, Finland. Phone: 358-9-708 59 411. Fax: 358-9-708 59 416. E-mail: Panu.Hendolin{at}Helsinki.fi.

Journal of Clinical Microbiology, January 2000, p. 125-132, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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