Objective     To select and optimize the PCR buffer for Borna disease virus real-time fluorescence quantitative PCR diagnostic kit in order to significantly improve PCR amplification efficiency to BDV p24 gene.     Methods     Conventional PCR was used to contrast the PCR buffers which titrated with hydrochloric acid

phosphoric acid and sulfuric acid respectively

and the appropriate concentration of ammonium sulfate was further explored to prepare TSS buffer. Four PCR additives (TMAC

Triton X-100

DMSO and betaine) were added into TSS buffer respectively to determine each optimal working concentration. One appropriate additive was mixed into other additives by turns in order to find out the most effective combination. Finally the efficiency of these different PCR buffers was verified by real-time PCR.     Results     Compared to other acids

sulfuric acid was more suitable to titrate the buffer. The optimal concentration of ammonium sulfate was identified as 6 mmol/L to prepare TSS buffer. Furthermore

real-time PCR results indicated that TBTT buffer (TSS buffer mixed with 1.5 mol/L betaine

8 mmol/L TMAC

and 0.15% Triton X-100) was the most effective buffer compare to other PCR additive mix and was more effective to amplify p24 gene than Takara PCR buffer.     Conclusion     The PCR buffer for amplifying BDV p24 gene is successfully optimized.