Establishment and application of SYBR Green I real-time fluorescence quantitative PCR for detection of Anguillid herpesvirus

China owns the largest eel culture industry in the world. Since 1990s, "mucus sloughing and hemorrhagic septicemia disease" has become one of the main epidemic diseases of cultured juvenile eel, with typical symptoms of "mucus sloughing", "red head" and "hemorrhagic septicemia", and high morbidity and mortality. The disease could also be found in adult eels with typical symptoms of "hemorrhagic septicemia", resulting in a huge number of deaths, and enormous economic losses to the industry. Cultivation experience showed that the disease might be caused by viral pathogen. We had Anguillid herpesvirus 1 (AngHV) isolated from the clinical samples of the disease. Further pathogenicity and epidemiological analysis showed that AngHV was the pathogenic agent of eel "mucus sloughing and hemorrhagic septicemia disease". A rapid and sensitive detection method for AngHV is of great significance for the early diagnosis, prevention and control of AngHV disease. Real-time fluorescence quantitative PCR (qPCR) method has been widely used in pathogen detection. To establish SYBR Green Ⅰ qPCR method for detection of AngHV, the sequence of AngHV ORF49 was amplified by PCR and cloned into pET-32a vector to construct the standard plasmid pET-32a-ORF49. Primers were designed according to ORF49 sequence, and a SYBR Green Ⅰ qPCR method was developed for AngHV detection using serially diluted standard plasmid as templates, then the sensitivity, repeatability, specificity and application effects of the method were evaluated. The results showed that the cycle threshold value(C_T) of the qPCR assay had a good linear relationship with the copy number of the standard plasmid, the correlation coefficient (R²) of the obtained standard curve was 0.999, and the amplification efficiency was 100.855%. The melting curve analysis showed that there was only one melting peak with Tm values of 88.34-88.95 °C. Amplication specificity analysis indicated that the method could specifically detect AngHV, and had no amplification of Cyprinid herpesvirus 3 (CyHV-3), Rana grylio virus (RGV) and Eel iridovirus (EIV). The coefficient of variation within and between groups was less than 1% and 2%, respectively, which indicated that the method was repeatable. The qPCR method could detect a minimum of 10 viral copies with higher sensitivity than conventional PCR method which has detection limit of 1 000 copies. Application analysis indicated that AngHV could be detected in the main tissues of AngHV infected Anguilla anguilla with 5.70×10⁷ copies in gill, 1.91×10⁶ copies in mucus, 1.31×10⁶ copies in fin, 3.31×10⁴ copies in heart, 1.75×10⁴ copies in intestine, 1.25×10⁴ copies in kidney, 6.51×10³ copies in skin, 6.26×10³ copies in muscle, 6.13×10³ copies in brain, 2.48×10³ copies in liver and 1.81×10³ copies in spleen. The amounts of virions in gills, fins and skin mucus were significantly higher than those in other tissues. The positive detection rate of the qPCR method for the 25 suspected Anguilla spp. "mucus sloughing and hemorrhagic septicemia disease" samples was 96%, while that of the conventional PCR method was only 76%. In conclusion, the established SYBR Green I qPCR method has high sensitivity, strong specificity and good repeatability, and can be used for rapid and quantitative detection of AngHV, which would be helpful for the prevention and control of Anguilla spp."mucus sloughing and hemorrhagic septicemia disease".