Analytical specificity and sensitivity determination of 16SrRNA gene based diagnostic polymerase chain reaction (PCR) for molecular detection of Coxiella burnetii

Coxiella burnetii, the cause of Q-fever, is a widespread zoonosis. Classical methods for its isolation are time consuming and risky, requiring biosafety level 3 laboratory. However, this limitation is omitted in molecular detection. The aim of this study was to evaluate the use of 16SrRNA gene in molecular detection of C. burnetii. For this purpose, specific primers were designed for this gene and a polymerase chain reaction (PCR) assay was prepared using these primers. To construct a positive control plasmid, the PCR product was cloned in pTZ57R/T vector. Sensitivity of the assay was determined using PCR in 10 fold serial dilutions of the positive control plasmid, and then, the last concentration with positive band was determined as the limit of detection (LOD) of the assay. Specificity of the test was assessed with PCR in genomic DNA of the negative control bacteria. The PCR results showed the presence of a 240 bp band as expected. Sensitivity assessment revealed that the LOD of the assay was 1 ng. No amplification was exhibited in negative control bacteria after PCR. These data proved the specificity of the procedure. It is concluded from the results of this study that 16SrRNA gene is an appropriated gene for detection of C. burnetii.   Key words: Coxiella burnetii, Q-fever, 16SrRNA gene, polymerase chain reaction (PCR).