Establishment of PCR with LNA Modified Primers for Detection of African Swine Fever Virus

doi:10.11924/j.issn.1000-6850.casb2020-0643

Abstract

Abstract:

In order to establish a simple, rapid and accurate method for detecting African swine fever virus, in this study a pair of specific primers were designed according to the p72 gene sequence of ASFV published in GenBank, and the appropriate bases in the primers were modified with locked nucleic acid. By optimizing the annealing temperature and the primers concentration, a PCR method with LNA modified primers for detecting ASFV was established. The results show that this method is highly sensitive and specific, and the sensitivity can reach 3×10¹ copies/uL, which is 100 times higher than that of PCR with conventional primers and 10 times higher than that of real-time PCR. The method has no amplification of the genomes of classical swine fever virus, porcine circovirus type 2 and pseudorabies virus, with good specificity. The results of 72 clinical samples are consistent with the results of qPCR recommended by OIE, and the coincidence rate is 100%. In this study, a LNA primer PCR method for detecting ASFV is established, with high specificity, sensitivity and simple operation, providing a novel, sensitive and specific detection technique for the detection of ASFV.

Key words: ASFV, p72 gene, LNA primer, PCR method

CLC Number:

S855.3

Li Yan, Zhang Yuxuan, Chu Pinpin, Jiang Zhiyong, Xi Zhenjun, Cai Rujian, Gou Hongchao, Zhang Kunli, Song Shuai, Bian Zhibiao, Li Chunling. Establishment of PCR with LNA Modified Primers for Detection of African Swine Fever Virus[J]. Chinese Agricultural Science Bulletin, 2021, 37(29): 107-113.

Figures/Tables 8

References 31

[1]	林祥梅, 韩雪清, 王景林, 等. 外来动物疫病[M]. 北京: 科学出版社, 2014:258-269.
[2]	王清华, 任炜杰, 包静月, 等. 我国首例非洲猪瘟的确诊[J]. 中国动物检疫, 2018, 35(9):1-4.
[3]	Zhou X, Li N, Luo Y, et al. Emergence of African swine fever in China[J]. Transbound Emerg Dis, 2018, 65(6):1482-1484. doi: 10.1111/tbed.2018.65.issue-6 URL
[4]	Alejo A, Matamoros T, Guerra M, et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92(23):1681-1690.
[5]	Dixon L K, Chapman D A G, Netherton C L, et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173(1):3-14. doi: 10.1016/j.virusres.2012.10.020 URL
[6]	Alonso C, Borea M, Dixon L, et al. ICTV virus taxonomy profile: Asfarviridae[J]. J Gen Virol, 2018, 99(5):613-614. doi: 10.1099/jgv.0.001049 URL
[7]	Wang AP, Jia R, Liu YC, et al. Development of a novel quantitative real-time PCR assay with lyophilized powder reagent to detect African swine fever virus in blood samples of domestic pigs in China[J]. Transboundary and Emerging Diseases, 2019, 00:1-14.
[8]	OIE. Terrestrial animal health code. Chapter 2.8.1.African Swine Fever [DB]. 2012.
[9]	贾云飞, 赵福杰, 朱静静, 等. 非洲猪瘟病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立[J]. 河南农业大学学报, 2020, 54(1):69-80.
[10]	崔贝贝, 李霆, 仇松寅, 等. 基于E184L基因的非洲猪瘟病毒实时荧光定量PCR检测方法的建立[J]. 动物医学进展, 2020, 41(1):8-14.
[11]	Feng Z S, Zhao L, Wang J, et al. A multiplex one-tube nested real-time RT-PCR assay for simultaneous detection of respiratory syncytial virus,human rhinovirus and human metapneumovirus[J]. Virol J, 2018, 15:167.
[12]	Wang J, Zhao L, Sun Z H, et al. Development of an innovative one-step nested PCR strategy for virus detection using the LNA technique[J]. Sci China Life Sci, 2019, 62(3):428-430. doi: 10.1007/s11427-018-9347-1 pmid: 30519878
[13]	Zhang R Q, Li Z, Li G X, et al. A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique[J]. 2020, Int Infect Dis, 93:224-230.
[14]	Tan T T, Pawestri H A, My N N, et al. A real-time RT-PCR for detection of clade 1 and clade 2 H5N1 influenza A virus using locked nucleic acid (LNA) TaqMan probes[J]. Virology Journal, 2010, 7:46. doi: 10.1186/1743-422X-7-46 URL
[15]	Nafa K, Hameed M, Arcila M E. Locled nucleic acid probes (LNA)for enhanced detection of low-level, clinically significant mutations[J]. Methods Mol Biol, 2016, 1392:71-82.
[16]	边立忠, 宋广辉, 赵素芝, 等. LNA探针实时荧光定量PCR检测HBV DNA的研究[J]. 中华临床医师杂志, 2013, 7(6):2501-2506
[17]	World Organisation for Animal Health(OIE). OIE Terrestrial Manual2012:Chapter2.8.1,African swine fever [EB/OL]. [2018-09-22]. pp.1067-1081. http://120.52.51.14/www.oie.int/fileadmin/Home/eng/Heaothstandards/tahm/2.08.01-ASF.pdf.
[18]	闫晓明. 非洲猪瘟疫情对生猪产业链上市公司证券市场价格影响的实证研究[J]. 中国农学通报, 2019, 35(34):160-164.
[19]	王彩霞, 吴绍强, 王芹, 等. 非洲猪瘟病毒CD2v基因实时荧光PCR检测方法的建立[J]. 中国兽医科学, 2020, 50(9):1083-1089.
[20]	Wang Y, Xu LZ, NOLL L, et al. Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control[J]. Transboundary and Emerging Diseases, 2020, 00:1-9.
[21]	严礼, 宋晟, 张继红, 等. 非洲猪瘟病毒微滴式数字PCR检测方法的建立与应用[J]. 激光生物学报, 2020, 29(4):344-351.
[22]	Wu XL, Xiao L, Lin H, et al. Development and application of a droplet digital polymerase chain reaction (ddPCR) for detection and investigation of African swine fever virus[J]. Canadian Jounal of Veterinary Research, 2018, 82(1):70-74.
[23]	王林, 高晓龙, 吴迪, 等. 非洲猪瘟病毒实时荧光LAMP检测方法的建立与应用[J]. 中国兽药杂志, 2020, 54(8):1-8.
[24]	Wang D G, Yu J H, Wang Y Z, et al. Development of a real-time loop-mediated isothermal amplication (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV)[J]. 2020, 276,113775-1137781.
[25]	Donnell V K, Grau F R, Mayr G A, et al. Rapid sequence-based characterization of African swine fever virus by use of the oxford nanopore minion sequence sensing device and a companion analysis software tool[J]. Journal of clinical microbiology, 2019, 58,1
[26]	姜睿姣, 张鹏飞, 朱光恒, 等. 非洲猪瘟检测技术进展[J]. 病毒学报, 2019, 35(3):523-531.
[27]	Luo Y Z, Atim S A, Shao L N, et al. Development of an updated PCR assay for detection of African swine fever virus[J]. Arch Virol, 2017, 162(1):191-199. doi: 10.1007/s00705-016-3069-3 URL
[28]	Zhao L, Wang J, Li G X, et al. A highly sensitive 1-tube nested real-time RT-PCR assay using LNA modified primers for detection of respiratory syncytial virus[J]. Diagn Microbiol Infect Dis. 2019, 93(2):101-106. doi: 10.1016/j.diagmicrobio.2018.09.001 URL
[29]	张晓勇, 罗前程. 应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变[J]. 中国生物工程杂志, 2018, 38(9):48-54.
[30]	陈茹, 段燕喻, 高小博, 等. 建立多重液相基因芯片方法快速检测狐狸、水貂成分[J]. 中国生物工程杂志, 2019, 39(11):62-69.
[31]	李艳, 楚品品, 雷雯, 等. 猪伪狂犬病毒野毒LNA探针实时荧光定量PCR检测方法的建立[J]. 中国动物传染病学报, 2021, 29(1):36-41.

引物名称	序列(5＇-3＇)	产物大小
P1	GGTAATGTGATCGGATACGTAACG	147 bp
P2	TTGGCACAAGTTCGGACATGTTGT
P3	GGTAATGTGAtCGGaTACGtAACG
P4	TTGGCACAaGTtCGGaCATGTTGT

引物名称	序列(5＇-3＇)	产物大小
P1	GGTAATGTGATCGGATACGTAACG	147 bp
P2	TTGGCACAAGTTCGGACATGTTGT
P3	GGTAATGTGAtCGGaTACGtAACG
P4	TTGGCACAaGTtCGGaCATGTTGT

检测结果	LNA引物 PCR	OIE推荐real-time PCR
阳性数	0	0
阴性数	72	72
总计	72	72

检测结果	LNA引物 PCR	OIE推荐real-time PCR
阳性数	0	0
阴性数	72	72
总计	72	72