Development of a Quantitative SYBR Green Real time PCR assay for specific detection of
Coxiella burnetii
Introduction and Objectives: Coxiella burnetii is the causative agent of Q-fever, a worldwide
zoonosis that has been categorized in biosafety level 3 agents. We developed a quantitative SYBR
Green real time PCR assay for rapid detection of the bacterium.
Materials and methods: 16srRNA gene was targeted and specific primers were designed using
primer BLAST of NCBI. The SYBR Green Real time PCR assay was established. The test specificity
was evaluated using the other bacterial genomes. Construction of positive control plasmid was carried
out with cloning of the PCR product into pTZ57R/T vector. The sensitivity of the assay was tested by
performing the assay on the 10 fold serial dilution of the positive control plasmid with initial
concentration of 200 ng/µl. To develop the assay as a quantitative test, the standard curve was
depicted based on Ct value of the serial dilutions against to the concentrations.
Results: Specific amplification of 16srRAN gene of C. burnetii was occurred in Tm of 85ºC as
expected. Result of the amplification in specificity testing was negative showing the specificity of the
designed assay. The limit of detection of the assay was 20 pg. After plotting the standard curve, R2,
slope and efficiency of the standard curve calculated as 0.99, 3.4 and 98% respectively. The linearity
of the quantitative assay was in the range of 200 ng – 20 pg of the positive control plasmid.
Conclusion: The results showed high specificity and sensitivity of the SYBR Green Real-time PCR
for rapid and quantitative detection of C. burnetii.
Keywords: Coxiella burnetii, diagnosis, SYBR Green Real-time PCR.