关键词: 非洲猪瘟病毒(AFSV); 假病毒; 阳性对照品

Abstract: 【Objective】 The purpose of this study was to prepare a safe and stable quantitative Real-time PCR positive control for African swine fever virus (ASFV).【Method】 The ASFV nucleotide sequence containing P72, CD2 v and MGF360-12 L gene fragments was synthesized,and the three gene segments were inserted in series into the Adenovirus vector PacAd5.The recombinant Adenovirus plasmid and Adenovirus skeleton plasmid were co transfected into 293A cells,and the expression of green fluorescent protein (EGFP) was observed by inverted fluorescent microscope.After 10 days of culture,the pseudovirus packaging was detected by PCR.The prepared pseudovirus particles were added with freeze-dried protective agent to prepare a positive control,and the homogeneity and stability tests were carried out,and the prepared positive control was subjected to the absolute quantification of the number of nucleic acid copies.【Result】 The 293A cells co-transfected with recombinant Adenovirus plasmid and Adenovirus skeleton plasmid showed spot fluorescence 24 h later,the cells showed a tendency to form island like infection area 7 days after transfection,and the cells showed obvious cytopathic effects such as pyknosis and abscission 10 days after transfection.The harvested pseudovirus liquid was detected by PCR and sequenced,and the result showed that it could amplify about 2 400 bp positive band,and the sequencing sequence was consistent with the inserted fragment.The homogeneity test showed that the coefficient of variation of Ct value of the positive control was less than 1%,indicating good homogeneity.Accelerated thermal stability test showed that the prepared positive control sample remained stable after being placed at 4 ℃ for 7 days,treated at room temperature (25 ℃) and 37 ℃ for 24 h respectively.The positive control still had good stability after DNase Ⅰ treated for 1 h.The storage period test showed that the prepared positive control could be stored stably for 6 months at －20 ℃.The prepared positive control was detected by ddPCR,and the result showed that the nucleic acid copy number of the prepared positive control pseudovirus solution was (2.26±0.09)×10⁴ copies/μL.【Conclusion】 The prepared ASFV pseudovirus positive control containing P72, CD2 v and MGF360-12 L gene fragments by using lentiviral packaging technology in this study were constructed successfully,which could be used for DNA extraction and molecular detection.

Key words: African swine fever virus (AFSV); pseudovirus; positive control