猪瘟病毒巢式RT-PCR检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2016.09.010

Abstract

Abstract:

A nested RT-PCR for detection of classical swine fever virus (CSFV) was developed using the total RNA of CSFV as a reverse transcription template,and two pairs of specific primers were designed based on the conserved region of CSFV E2 gene.The specificity,sensitivity and repetition of CSFV nested RT-PCR were tested,and 35 samples taken from clinical suspicious CSFV infected pigs were tested by the established nested RT-PCR assay.The results indicated that the nested RT-PCR assay was successfully established.The sensitivity and specificity of the nested RT-PCR assay revealed that the minimum detectable virus content limit was 1 TCID₅₀,and no products were amplified from the nucleic acid of ST normal cell or other 8 pathogenic viral or bacterial microorganisms.The repetition test indicated the nested RT-PCR assay showed a good repeatability.Meanwhile,19 positive samples were detected in 35 suspected samples,which was consistent with the results tested by sequencing.The study suggested that the established nested RT-PCR assay was specific,sensitive and suitable for early detection of CSFV.

Key words: classical swine fever virus; nested RT-PCR; detection; establishment; application

CLC Number:

S852.65⁺1

LIU Mei-fen, WANG Shu-juan, YAN Ruo-qian, WANG Dong-fang, CAO Wei-wei, ZHAO Xue-li, LI Qin-nan, LI Ning. Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus[J]. , 2016, 43(9): 2285-2290.

References

[1] Van Oirschot J T.Vaccinology of classical swine fever:From lab to field[J].J Vet Microbiol, 2003,96(4):367-384.
[2] Francki R I B,Fauquet C,Knudson D,et al.Classification and nomenclature of viruses[C].Fifth report of the International Committee on Taxonomy of Viruses,1991,6:119-138.
[3] Schirrmeier H,Strebelow G,Depner K,et al.Genetic and antigenic characterization of an atypical pestivirus isolate,a putative member of a novel pestivirusspecies[J].Journal of General Virology, 2004,85(12):3647-3652.
[4] Harris E,Holden K L,Edgil D,et al.Molecular Biology of Flaviviruses[M].New Treatment Strategies For Dengue and other Flaviviral Diseases,2006.
[5] 周绪斌,王新平,宣华,等.鉴别牛病毒性腹泻粘膜病毒和CSFV复合PCR方法及其应用[J].中国兽医学报,2002,22(6):173-179.
[6] 李晶梅,刘丹,薛霜,等.猪瘟病毒和牛病毒性腹泻病毒双重RT-PCR鉴别检测方法的建立及应用[J].动物医学进展,2014,35(4):66-71.
[7] 谢金文,李安,肖跃强,等.采用SYBR GreenⅠ实时荧光定量RT-PCR方法对猪瘟强毒与疫苗弱毒的鉴别研究[J].中国预防兽医学报,2012,34(1):45-48.
[8] 邓力,张彦明,李维维,等.猪瘟兔化弱毒荧光定量PCR检测方法的建立及初步应用[J].西北农林科技大学学报,2011,39(2):1-8.
[9] 范斌,许文花,韩建强,等.猪瘟兔化弱毒疫苗中病毒含量实时荧光定量RT-PCR检测方法的建立及初步应用[J].中国畜牧兽医,2014,41(12):56-61.
[10] Haegeman A,Dewulf J,Vrancken R,et al.Characterisation of the discrepancy between PCR and virus isolation in relation to classical swine fever virus detection[J].J Virol Methods,2006,136(1-2):44-50.
[11] Handel K,Kehler H,Hills K,et al.Comparison of reverse transcriptase-polymerase chain reaction,virus isolation,and immunoperoxidase assays for detecting pigs infected with low,moderate,and high virulent strains of classical swine fever virus[J].J Vet Diagn Invest,2004,16(2):132-138.
[12] Dewulf J,Koenen F,Mintiens K,et al.Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection[J].J Virol Methods,2004,119(2):137-143.
[13] Moennig V,Floegel-Niesmann G,Greiser-Wilke I.Clinical signs and epidemiology of classical swine fever:A review of new knowledge[J].Vet J,2003,165(1):11-20.
[14] Sand-Vik T,Paton D J,Lowings P J.Detection and identification of ruminant and porcine pestiviruses by nested amplification 5'untranslated coding regions[J].J Virol Methods,1997,64(1):43-56.
[15] Goldrick M A,Lowings J P,Ibala G,et al.A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorgenic probe (Taqman)[J].J Virol Methods,1998,72(2):125-135.
[16] Yu M,Wang L F,Shiell B J,et al.Fine mapping of a C-term inal linear epitope highly conserved among the major enevelope glycoprotein E2(gp51 to gp54) of different pestviruses[J].Virology, 1996,222(1):289-292.
[17] 刘俊,王琴,范学政,等.猪瘟病毒野毒株TaqMan-MGB荧光定量PCR鉴别方法的建立与应用[J].中国农业科学,2009,42(12):4366-4371.
[18] 赵建军,成丹,李娜,等.猪瘟病毒实时荧光定量RT-PCR的建立及其对人工感染猪体内猪瘟病毒的检测[J].畜牧兽医学报,2007,38(7):685-693.
[19] Handel K,Kehler H,Hills K,et al.Comparison of reverse transcriptase-polymerase chain reaction,virus isolation,and immunoperoxidase assays for detecting pigs infected with low,moderate,and high virulent strains of classical swine fever virus[J].J Vet Diagn Invest, 2004,16(2):132-138.
[20] 吴鑫,王军,张以芳.猪瘟病毒一步法RT-PCR检测方法的建立[J].中国畜牧兽医,2008,35(2):64-66.

Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus

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