用于非洲猪瘟病毒核酸检测的假病毒阳性对照品的制备和标定

doi:10.16431/j.cnki.1671-7236.2023.02.022

Abstract

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

CLC Number:

S852.65^＋1

LI Pengfei, SONG Xiaoming, ZHOU Baokun, CAO Zhi, ZHANG Hongliang, MA Qingxia, SHAN Hu. Preparation and Calibration of Pseudoviral Positive Control for Nucleic Acid Detection of African Swine Fever Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 647-655.

References

[1] 吴萌萌,张栋良,孙彩虹,等.非洲猪瘟研究进展[J].畜牧兽医杂志,2022,41(1):43-47. WU M M,ZHANG D L,SUN C H,et al.Research progress of African swine fever[J]. Journal of Animal Science and Veterinary,2022,41(1):43-47.(in Chinese)
[2] ZHOU X,LI N,LUO Y,et al.Emergence of African swine fever in China,2018[J]. Transboundary and Emerging Diseases,2018,65(6):1482-1484.
[3] SALAS M L,ANDRES G.African swine fever virus morphogenesis[J]. Virus Research,2013,173(1):29-41.
[4] GALINDO I,ALONSO C.African swine fever virus:A review[J]. Viruses,2017,9(5):103.
[5] BORCA M V,IRUSTA P,CARRILLO C,et al.African swine fever virus structural protein p72 contains a conformational neutralizing epitope[J]. Virology,1994,201(2):413-418.
[6] O’DONNELL V,HOLINKA L G,GLADUE D P,et al.African swine fever virus Georgia isolate harboring deletions of MGF360 and MGF505 genes is attenuated in swine and confers protection against challenge with virulent parental virus[J]. Journal of Virology,2015,89(11):6048-6056.
[7] LI D,LIU Y,QI X,et al.African swine fever virus MGF-110-9L-deficient mutant has attenuated virulence in pigs[J]. Virologica Sinica,2021,36(2):187-195.
[8] RAMIREZ-MEDINA E,VUONO E,O’DONNELL V,et al.Differential effect of the deletion of African swine fever virus virulence-associated genes in the induction of attenuation of the highly virulent Georgia strain[J]. Viruses,2019,11(7):599.
[9] 程晶,刘文晓,王晓玥,等.非洲猪瘟诊断方法研究进展[J].中国畜牧兽医,2022,49(5):1985-1993. CHENG J,LIU W X,WANG X Y,et al.Research progress on development of diagnostic methods of African swine fever[J]. China Animal Husbandry & Veterinary Medicine,2022,49(5):1985-1993.(in Chinese)
[10] BERND H,MARTIN B,SCOTT M R,et al.A review of RT-PCR technologies used in veterinary virology and disease control:Sensitive and specific diagnosis of five livestock diseases notifiable to the World Organization for Animal Health[J]. Veterinary Microbiology,2009,139(1-2):1-23.
[11] LIN Y,CAO C,SHI W,et al.Development of a triplex Real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus[J]. Journal of Virological Methods,2020,280:113875.
[12] 韩勇军,曹鹏程,赵亚齐,等.鉴别检测野生株和基因缺失株非洲猪瘟病毒多重荧光定量PCR方法的建立[J].中国兽医科学,2022,52(1):41-47. HAN Y J,CAO P C,ZHAO Y Q,et al.Establishment of a multiplex Real-time PCR method for differential detection of wild strains and gene-deleted strains of African swine fever virus[J]. Chinese Veterinary Science,2022,52(1):41-47.(in Chinese)
[13] 郭振华,邢广旭,翁茂洋,等.非洲猪瘟病毒野毒株与基因缺失株三重荧光定量PCR鉴别检测方法的建立及应用[J].中国兽医学报,2022,42(4):619-625. GUO Z H,XING G X,WENG M Y,et al.Development and application of a triplex Real-time PCR method for detection and differentiation of gene-deleted and wide-type African swine fever virus[J]. Chinese Journal of Veterinary Science,2022,42(4):619-625.(in Chinese)
[14] 世界动物卫生组织OIE.OIE陆生动物诊断试验与疫苗手册-哺乳动物、禽类与蜜蜂[M].北京:中国农业出版社,2017. World Organization for Animal Health.OIE Handbook of Terrestrial Animal Diagnostic Tests and Vaccines -Mammals,Birds and Bees[M].Beijing:China Agriculture Press,2017.(in Chinese)
[15] SANCHEZ R O,GONZALEZ P A,GOMEZ-PUERTA S,et al.Avian CD154 enhances humoral and cellular immune responses induced by an Adenovirus vector-based vaccine in chickens[J]. Comparative Immunology, Microbiology and Infectious Diseases,2011,34(3):259-265.
[16] RAUSCHHUBER C,NOSKE N,EHRHARDT A.New insights into stability of recombinant Adenovirus vector genomes in mammalian cells[J]. European Journal of Cell Biology,2012,91(1):2-9.
[17] POULIN K L,MCFALL E R,CHAN G,et al.Fusion of large polypeptides to Human adenovirus type 5 capsid protein Ⅸ can compromise virion stability and DNA packaging capacity[J]. Journal of Virology,2020,94(17):e01112-20.
[18] BETT A J,PREVEC L,GRAHAM F L.Packaging capacity and stability of Human adenovirus type 5 vectors[J]. Journal of Virology,1993,67(10):5911-5921.
[19] HARUI A,SUZUKI S,KOCHANEK S,et al.Frequency and stability of chromosomal integration of Adenovirus vectors[J]. Journal of Virology,1999,73(7):6141-6146.
[20] KETZER P,HAAS S F,ENGELHARDT S,et al.Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic Adenoviruses[J]. Nucleic Acids Research,2012,40(21):e167.
[21] 周冬根,李云鹏,罗洁,等.用于MERS-CoV核酸检测的假病毒阳性对照品制备及鉴定[J].中国国境卫生检疫杂志.2019,42(4):233-238. ZHOU D G,LI Y P,LUO J,et al.Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-COV[J]. Chinese Journal of Frontier Health and Quarantine,2019,42(4):233-238.(in Chinese)
[22] 郑夔,袁帅,黎东红,等.新疆出血热病毒实时荧光RT-PCR检测方法的假病毒阳性对照品制备[J].华南预防医学,2016,42(1):1-5. ZHENG K,YUAN S,LI D H,et al.Preparation of pseudoviral positive control for Real-time RT-PCR detection of Xinjiang Hemorrhagic fever virus[J]. South China Journal of Preventive Medicine,2016,42(1):1-5.(in Chinese)
[23] 戚宇华,崔仑标,史智扬,等.含H5N1禽流感病毒核酸序列的病毒样颗粒的制备及应用[J].中国人兽共患病学报.2010,26(1):29-32. QI Y H,CUI L B,SHI Z Y,et al.Preparation of the RNase-resistant virus particles containing the partial gene fragments of Avian influenza virus H5N1 and its application[J]. Chinese Journal of Zoonoses,2010,26(1):29-32.(in Chinese)

Preparation and Calibration of Pseudoviral Positive Control for Nucleic Acid Detection of African Swine Fever Virus

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