Establishment of a TB Green Real Time Fluorescent Quantitative PCR for Duck hepacivirus
CHEN Cui-Teng, WAN Chun-He*, CHEN Zhen, ZHU Chun-Hua, LIU Bin-Qiong, CAI Guo-Zhang, HUANG Yu*
Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention/Fujian Animal Diseases Control Technology Development Center, Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
Abstract：Duck hepacivirus (DuHCV) is a newly identified Hepatitis C virus in ducks (Anas) with abnormal egg production syndrome. In order to establish a molecular biological detection method of DuHCV for the epidemiological investigation of the disease and further explore its pathogenic mechanism. In this study, based on the NS5B gene characteristics of the DuHCV reference strain (HCL-2 strain, GenBank No. MK737640.1), specific primers were designed to establish a TB Green real-time fluorescent quantitative PCR method for the detection of DuHCV. The established TB Green real-time fluorescent quantitative PCR method of DuHCV had high sensitivity, with the limit of detection was 64 copies/μL. It had good specificity, with no cross-reaction with common duck origin infectious disease pathogens (such as Duck hepatitis A virus type 1, Duck hepatitis A virus type 3, Avian influenza virus, Avian paramyxovirus type 1, Avian tembusu virus, Muscovy duck reovirus and novel Muscovy duck reovirus), and the melting curve appeared a specific peak, with Tm value was (82.22±0.21) ℃. It had good reproducibility, and the coefficient of variation of repeated experiments within and between groups was 0.13%~0.54% and 0.53%~1.12%, respectively. 103 samples came from three species of ducks, Muscovy ducks (Cairna moschata), Jinding ducks and Putian black ducks were collected, and then were tested with the established TB Green real-time fluorescent quantitative PCR method and reverse transcription PCR (RT-PCR) method simultaneously for DuHCV infection. The results showed that 8 positive (positive rate was 7.8%) samples were detected by the RT-PCR method, while the TB Green real-time fluorescent quantitative PCR method had 11 positive (positive rate was 10.68%) samples. Moreover, 8 RT-PCR positive samples were all positive by TB Green real-time fluorescent quantitative PCR method, with the coincidence rate was 100%. DuHCV positive was not detected in Muscovy ducks, but both DuHCV positive were found in Jinding ducks and Putian black ducks. Previously data showed no reports of Putian black ducks infected with DuHCV, which means this study confirmed the existence of DuHCV firstly. In conclusion, TB Green real-time fluorescent quantitative PCR method for the detection of DuHCV was successfully established, with high specificity, sensibility and well repetitiveness, which provides a useful candidate tool for epidemiological investigation and pathogenic mechanism research of DuHCV.
陈翠腾, 万春和, 陈珍, 朱春华, 刘斌琼, 蔡国漳, 黄瑜. 鸭丙型肝炎病毒TB Green实时荧光定量PCR方法的建立[J]. 农业生物技术学报, 2021, 29(12): 2465-2472.
CHEN Cui-Teng, WAN Chun-He, CHEN Zhen, ZHU Chun-Hua, LIU Bin-Qiong, CAI Guo-Zhang, HUANG Yu. Establishment of a TB Green Real Time Fluorescent Quantitative PCR for Duck hepacivirus. 农业生物技术学报, 2021, 29(12): 2465-2472.
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