Use of multiplex PCR fur simultaneous detection of four bacterial species in middle ear effusions

A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenza, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion, The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers, The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products, The specificity of the reaction was verified Ft-il-h various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods, A detection level of 10 bacterial, cells/reaction for each or the study organisms was achieved, By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms, In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCB negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms, Thus, the multiplex PCB method improves the detection rate significantly compared to that of the conventional culture method.