Abstract:?Objective?To construct a fuse gene of the fragments from five Rickettsiae by two overlapping extension PCR (SOE-PCR) methods, and compared the results of the two methods.?Methods?The fused genes from five Rickettsiae were constructed by one-step and two-step fusion gene techniques respectively, and the concentration of overlapping primers in the one-step method and the addition amount of target gene products in the two-step method were optimized, so as to improve the fusion efficiency of the two methods.?Results?A fused gene from five Rickettsiae was successfully established. In the process of one-step gene fusion, when the concentration of overlapping primers was 0.01 to 0.1 μmol/L, the band of 912 bp fusion gene had higher brightness and fewer miscellaneous bands, while in the two-step gene fusion process, when the amount of target gene products was 0.1 to 0.25 μL, the band of 912 bp fusion gene had higher brightness and fewer miscellaneous bands.?Conclusion?The one-step gene fusion technology should be an economical, convenient and efficient fusion technology since its simple operation, short time consumption and less consumptive materials under the conditions of controlling the temperature and time of annealing. It may provide an experimental template for the subsequent construction of Rickettsiae fusion gene expression vector and multiple detections of Rickettsiae with molecular biological methods.