Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Haemophilus Influenzae Bacterium in Clinical Sample of Sinusitis

Haemophilus influenza is a human-restricted Gram-negative bacterium that is part of the normal nasopharyngeal flora of most humans. H. influenzae can cause diseases such as pneumonia, meningitis, bacteraemia, sinusitis and acute otitis media. Nonculture methods like PCR have become available during the last two decades, providing early and accurate diagnosis of bacterial diseases. The purpose of this study was to design a PCR assay for the rapid detection of H. influenzae bacterium. In this study the target gene was P6 so the specific primers were designed for it. The PCR reaction was set up on the DNA genomic of H. influenzae. To create a positive control, the PCR product was cloned in the pTZ57R/T vector and was transformed into E. coli JM107. The sensitivity of this assay was tested by a serial dilution of the positive control. A total of 72 clinical samples collected from patients with sinusitis were analyzed by PCR. PCR results showed a band of the expected size 273 bp. Sensitivity results indicated that the limit of detection of the assay was 1 CFU/ml. No amplification was observed after PCR with any of the microorganisms tested. There was 19 (26%) positive numbers of H. influenza in the samples in this study. The PCR method showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes.