The high quality hot start Taq DNA polymerase used in this assay could minimize unspecific amplifications and increase the PCR cycling efficiency. In addition, FQ-PCR reaction and detection is all done in a closed-tube system, the need for post-amplification manipulation is removed since the detection of the PCR products occurs online during real-time PCR amplification, hence greatly reducing the risk of cross-contamination and false positive results. The optimization of the AHV-1 FQ-PCR assay was focused on the concentration of primers and probe and Mg 2+ . When all these different practical refinements are combined, the final result is a molecular diagnostic method that is not only rapid and reliable, but one that is also easy to perform and applicable to use for testing large numbers of samples since the FQ-PCR presented the benefits of increased speed due to reduced cycle time and remove of post-amplification process, offering considerable labor savings and allowing higher throughput analysis than conventional PCR assays and thus is favorable for the transition of this method from research to routine use in laboratories. This method was preliminarily mentioned in a short report [26] but related details of primers and probe sequence, specificity test, sensitivity test, reproducibility analysis, dynamic range and internal control were unavailable. By contrast, great modification and optimization have been made in this paper to improve the quality of this study.