A Real-time Quantitative Loop Mediated Isothermal Amplification Assay Targeting com1 Gene of Coxiella burnetii

Abstract

Background & Objectives: The present study designed a loop mediated isothermal amplification (LAMP) method to rapidly detect Coxiella burnetii (C. burnetii) and develop a sensitive Real-time quantitative LAMP (Q-LAMP) assay to quantify Q fever. Materials & Methods: Primers were specifically designed for use in targeting the com1 conserved gene of C. burnetii to carry out LAMP detection of the agent causing Q fever. After obtaining the LAMP reaction, the sensitivity of the method was assessed by preparing a serial ten-fold dilution of a plasmid carrying com1 gene. Results: The assay’s sensitivity for the visual detection of changes in turbidity or turbidimeter and electrophoresis of agarose gel were 0.4 fg and 0.04 fg, respectively. Hence, the lower limit of detection was 120 and 12 copies of the gene detected in 60 min. The results of this study were indicative of the simplicity, rapidness, sensitivity, and specificity of the LAMP assay for C. burnetii detection and a probable improvement of the diagnostic procedure used in clinical laboratories. Conclusion: The assay specificity was assessed using Coxiella genomic DNA and a panel of Gram-negative and Gram-positive bacteria. The LAMP assay was shown to be highly specific for detection of Coxiella without any observable amplification products from non-Coxiella organisms.

Keywords

• coxiella burnetii
• q fever
• loop mediated isothermal amplification (lamp)