Design of Polymerase Chain Reaction (PCR) Assay for Influenzae Bacterium Molecular Detection of HaemophilusInfluenzae Bacterium

Haemophilus influenza (H. influenza) is an important cause of meningitis in infants and children aged less than five years. For this reason the early diagnosis of this bacterium is important. Studies have shown that molecular methods are specific tests for early dete-ction for this agent. The purpose of this study was to design a PCR assay for the rapid detection of H. influenzae bacterium.

In this study specific primers were designed based on ompp6 gene and polymerase chain reaction (PCR) assay was setup. To create the standard positive control، the PCR product was cloned in pTZ57R/T vector. The existence of the desired gene in the T-vector was confirmed by digestion and sequencing processes. Sensitivity of the PCR assay was determined by preparing a serial tenfold dilutions of the positive control plasmid with starting concentration of 11ng/µl. The Specificity of the assay was verified by using of PCR on the genomic DNA of a variety of bacteria.

PCR results showed a band of the expected size 280bp. Sensitivity results indicated that the limit of detection of the assay was 317 copy numbers. No amplify-ication was observed after PCR in negative control bacteria genomic DNA. This outc-ome proved the specificity of the PCR assay. Discussion &

The results of this study showed that the PCR assay is a rapid، highly sensitive and specific test for detection of H. influenzae bacterium.