Establishment Of Indirect ELISA Method To Detect Q Fever Coxiella Burnetii

Q fever is a worldwide natural focus zoonosis disease and anthropozoonosis caused by coxiella burnetii, which is an acidophilous and obligate intracellular gram-negative bacterium. Coxiella burnetii has strong infectivity with human and domestic animals. Because of the non-specificity of clinical symptoms, the mistake diagnose and missed diagnose were usually occured and it made some difficulties on the clinical diagnosis and prevention of Q fever. So it is significant to establish a sensitive, specific and benefit method for large scale screening of detecting coxiella burnetii.Because of the phenomenon of the phase variation, there are two phases of the coxiella burnetii, the phase I of the virulent strain and the phase Ⅱ of the attenuated strain. There are no obvious difference of antigen diagnostic between phase Ⅰ and phase Ⅱ. The phase Ⅱ antibody could be induced by the phase Ⅰ in the early infection and the phase Ⅰ antibody could be produced in the late infection. So the phase Ⅱ antigen can react with the antibody of early period serum. In order to reduce the risk during the preparation and detection of antigen, we treated it as the antigen in our experiment.In this study, the whole bacteria protein of the coxiella burnetii attenuated strain phase Ⅱ was used as the antigen, the indirect ELISA to detect Q fever was then established. Through optimizing the concentration and the reaction condition, the best concentration of the coated phase Ⅱ antigen is7.8ug/mL, and the best coated condition is to stay overnight in4℃. The confining liquid is1%casein and the blocking condition is37℃for60min. The diluent of the serum is1%casein, the dilution ratio is1:400and the reaction condition is37℃for30min. The multiple dilution of the anti-bovine HRP IgG is1:5000, the diluent is1%casein and the reaction condition is37℃for30min.The time of the color reaction is15min and the stop buffer is0.5mol/L H2SO4Furthermore, the critical value of this method is0.44, the intra-and inter-batch variation coefficients are1%-4.5%and1.9%-8.2%respectively, and then the sensitivity and the specificity are better. A total of393serum samples from bovine were tested by ELISA, the result showed that the positive serums are45and the serum positive detection rate is11.45%. The same batch of the serums was tested by IDEXX Q-FEVER (Coxiella burnetii) Antibody Test Kit, the positive serum of the test result is24, and the serum positive detection rate is6.1%. Comparing the two detection methods, the coincidence is94.66%. It demonstrates that the sensitivity and the specificity of the established indirect ELISA detection method are better and could be used for the clinical diagnosis of the antibody of the coxiella burnetii.