An internal positive control was introduced into the FQ-PCR assay to verify the absence of DNA losses during the extraction step and of PCR inhibitors in the DNA templates. The internal positive control of pGM-T recombinant vector (designed as pB16S) consisting of Bacillus 16S rRNA gene (GenBank Accession No. AJ971894) sequence amplified with primers (IC-F and IC-R) listed in Table 3 was added into the lysis buffer at the concentration of 1.0 × 10 6 copies/μL. Real-time PCR for IC detection was carried out in a separate run, using primers and probe (named IC-F, IC-R and IC-P respectively) listed in Table 3 . The fluorogenic probe was labelled at 5' with FAM as reporter and labelled at 3' with TAMRA. The quantitative real-time PCR protocol was the same as that of AHV-1 detection. From the ratio of the calculated amount of IC to the actual amount of IC, which is shared by the specimen, the normalization could be achieved and the actual amount of AHV-1 in the specimen could be obtained. Actually this internally controlled method has been widely used in other related detection assays [28, 29] .