宿主DNA对埃立克体病PCR诊断的抑制作用研究

Study on inhibition of PCR for diagnosis of ehrlichiosis by host DNA

目的：优化埃立克体病的PCR诊断方法。方法：构建含有查菲埃立克体16S rRNA基因片段的重组载体。比较巢式PCR和普通一步法PCR的灵敏度差异。用RAW 264.7细胞DNA模拟实验室诊断时的宿主细胞DNA，评估宿主细胞DNA对巢式PCR效率的影响。添加BSA作为优化剂，探讨BSA是否能降低宿主DNA的干扰。结果：巢式PCR可以扩增出特异性产物的最低模板量为10^3拷贝，一步法PCR是10^5拷贝。50-100 ng的宿主细胞DNA能够降低巢式PCR的效率。BSA可以降低宿主细胞DNA对PCR的抑制作用。结论：巢式PCR的灵敏度明显高于一步法PCR；BSA可以降低宿主细胞DNA干扰，提高PCR的效率。建议埃立克体病的诊断应尽量选择巢式PCR，必要时可加入适量的BSA以提高灵敏度。

Objective：To optimize the PCR diagnosis of ehrlichiosis. Methods：A recombined plasmid containing a 16S rDNA fragment of E. chaffeensis was constructed. The sensitivity between nested PCR and normal one step PCR was compared. The DNA of RAW264. 7 was added to simulate the host cell DNA in the laboratory diagnosis, so that the interference of host DNA was evaluated. BSA was added as a component of PCR reaction, thus it was focused that whether the sensitivity of nested PCR could be affected and whether the interference from host cell DNA could be attenuated. Results：The lowest template concentration was 103 copies/reaction for nested PCR, while it was 105 copies/reaction for the one step PCR. The efficiency of nested PCR was reduced by 50 - 100 ng of host cell DNA. BSA could attenuate the inhibition by host cell DNA. Conclusion：The sensitivity of nested PCR is higher than that of one step PCR. BSA could enhance PCR by reducing the inhibition from host DNA. It is recommended by our results that nested PCR is prior to one step PCR in the diagnosis of ehrlichiosis, and BSA could be applied to increase the sensitivity of PCR if necessary.