A loop-mediated isothermal amplification for visual detection of Coxiella burnetti

Objective To develop a loop-mediated isothermal amplification (LAMP) assay for rapid and visual detection of Coxiella burnetti.Methods The recombinant plasmid containing IS1111a gene of Coxiella burnetti was constructed using in vitro gene synthesis.Three sets of LAMP primers targeting IS1111a gene were designed with Primer Explorer software 4.0.The most effective primer set was selected and the concentration of hydroxynaphthol blue (HNB) was optimized using real-time turbidimetry.Color change of the reaction tube was used to indicate the result.The detection limit of the optimized visual LAMP assay was evaluated using serially diluted recombinant plasmids of Coxiella burnetti.The specificity of the LAMP assay was tested using recombinant plasmid and XinQiao strain of Coxiella burnetti,together with other five members of genus Rickettsia.Results The optimized final concentration of HNB in the visual LAMP was 150 μM.The visual LAMP could specifically detect the recombinant plasmid and XinQiao strain of Coxiella burnetti,with no cross reactions with O.tsutsugamushi,Rickettsia rickettsii,spotted fever group Rickettsia,Rickettsia prowazekii,and Rickettsia Canada.The detection limit of this method was 3.6×10² copies/reaction,which was 10-fold higher than that of the conventional polymerase chain reaction (PCR),but was identical to that of real-time turbidimetry and quantitative real-time PCR (qPCR).The amplification time of the visual LAMP assay ranges from 20 to 40 minutes,much shorter than the 75 min for qPCR and 3 hours for conventional PCR.Conclusion The visual LAMP assay could detect Coxiella burnetti sensitively and specifically with less labor and time.It might be applicable for the routine surveillance of Coxiella burnetti in grassroots disease control and prevention institutes or in field test.