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Development Of An Evagreen-Based Real-Time Pcr Assay For Detection Of Aleutian Mink Disease Virus

文献类型： 外文期刊

作者： Li, L; Hu, Z; Sun, JH; Guo, K; Chu, XY; Wang, XJ; Lu, YX

作者机构：

关键词： Evagreen; Real-Time Pcr; Amdv; Ns Protein

期刊名称： JOURNAL OF VIROLOGICAL METHODS

ISSN： 0166-0934

年卷期： 2020 年 275 卷

页码：

收录情况： JCR（2021版）

摘要： The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8 degrees C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/mu L of AMDV plasmid or 3 pg/mu L of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

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