From the above results, the sensitivities of the first round of nested PCR with 24F1/24R1 or 25F1/25R1 were 5.6×104 and 1.1×104 copies of recombinant virus T-vector per 20 L PCR mixture, respectively. When the second round of PCRs was combined, the detection limit could be improved >1000 times. The sensitivity of ORF24 primers was better than ORF25, which was about 5.6 and 11 copies respectively. As the two primer sets have similar performance in detections, only ORF24 primer set was assessed in further sample detection