Rapid, accuracy assay for identifying of 3 pathogens such as Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis is needed in clinical practics. Multiplex PCR was established based on 3 known concentration strains of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis and diluted in a range of levels: 10 exponent 3
10 exponent 2
10 exponent 1
10 exponent 0 CFU/ml. The result showed that multiplex PCR can detect these pathogens with sensitivity and specificity are 1 CFU/ml and 97-100 percent, respectively.