关键词: 水貂阿留申病, 水貂阿留申病毒, 实时荧光定量PCR, VP2, 检测方法

Abstract:  Objective    To establish a convenient and sensitive fluorescent quantitative PCR method for the detection of Aleutian mink disease virus (AMDV), aim to implement the rapid quantitative detection of AMDV in clinical samples. Methods     According to the conserved region of VP2 gene of AMDV, a pair of specific primers and a probe were designed. The reaction condition was optimized, and the TaqMan real-time fluorescent quantitative PCR detection method was established. The standard curve was mapped, and the specificity, sensitivity and stability of the method were analyzed. Results    The correlation coefficient (r2) of the standard curve was more than 0.994, and it presented a linear relationship in the concentration of the template DNA ranging from 102 copies/µL to 108 copies/µL. The assay was highly specific for amplification from AMDV, but no amplification from CPV, MEV, CDV and CAV. The sensitivity of AMDV detection was 102 copies/µL, and the intra-group and inter-group reproducibility tests showed that the variation coefficient of the method was less than 3%. The result of 417 clinical tissue samples tested by this method showed that the positive rate of AMDV was 81.5% in some areas of China. Conclusion     A reproducible, specific and sensitive AMDV real-time PCR detection method is established, which can be used for clinical detection and epidemiological investigation of AMDV. 

Key words: Aleutian disease of mink, Aleutian mink disease virus, Real-time fluorescent quantitative PCR, VP2, Detection method