Abstract: This study aims to establish a high-efficiency, rapid, high-sensitivity and high-specificity TaqMan double real-time fluorescence quantification for duck hepatitis virus type 1 (DHAV-1) and duck hepatitis virus type 3 (DHAV-3) PCR (q-PCR) detection method and applied to the detection of clinical suspected samples. According to the conservative regions of the VP1 gene of DHAV-1 and DHAV-3, two pairs of specific primers and probes were designed and synthesized respectively. On this basis, the TaqMan dual real-time fluorescent quantitative PCR method was established and optimized. Results were as follows: The correlation coefficient (R²) of the optimized standard curve is above 0.999, the amplification efficiency (E) is between 105% and 110%, and the coefficient of variation (i-CV) and the coefficient of variation between groups (C-CV) are all below 0.77%. The double q-PCR method was used to detect mixed plasmids and viral nucleic acids of different dilutions. The results showed that the established TaqMan double q-PCR has high specificity; the sensitivity of simultaneous detection of DHAV-1 and DHAV-3 can reach 10 copies, Which are 1 000 times and 100 times higher than conventional PCR methods. Forty tissue samples of duck suspected of duck hepatitis virus infection from central Jiangsu and northern Jiangsu were tested. The results showed that 36 of them were positive by q-PCR method, with a detection rate of 90%; while conventional PCR methods failed to detect samples with high Ct values, and the detection rate was 72.5% (29 positive samples).The establishment of the TaqMan dual q-PCR detection method provides an efficient, sensitive, and specific tool for the detection of clinical samples of DHAV-1 and DHAV-3, and promotes clinical molecular epidemiological investigation and viral quantitative analysis.

Key words: duck hepatitis virus type 1, duck hepatitis virus type 3, duck viral hepatitis, real time fluorescent quantitative PCR