鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2021.018

摘要/Abstract

摘要： 旨在建立特异、敏感的实时荧光定量PCR（FQ-PCR）方法，用于非洲猪瘟病毒（ASFV）和猪瘟病毒（CSFV）野毒株的快速鉴别检测。针对ASFV的P72基因和CSFV野毒株的5'UTR非编码区序列的保守区域分别设计1对特异性引物和1条探针，经优化反应条件，建立一种基于TaqMan MGB探针技术的FQ-PCR方法，验证方法的敏感性、特异性和稳定性，对50份临床样品进行检测，并与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法进行比较分析。结果显示：建立的鉴别ASFV和CSFV野毒株二重FQ-PCR检测方法在10⁰~10⁶拷贝·μL^-1模板范围内有良好的线性关系；对ASFV和CSFV基因出现阳性扩增信号，但对猪瘟病毒疫苗株、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型、猪细小病毒、猪乙型脑炎病毒、副猪嗜血杆菌等病原对照未出现扩增；批内、批间试验变异系数在1.18%~2.08%，重复性良好；对ASFV和CSFV的最低检测模板浓度均为10拷贝·μL^-1；利用建立的二重FQ-PCR方法对50份临床样品进行检测，检测结果与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法结果完全一致。本研究成功建立了鉴别ASFV和CSFV野毒株二重TaqMan MGB FQ-PCR方法，为ASFV和CSFV野毒株的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。

关键词: 非洲猪瘟病毒, 猪瘟病毒野毒株, 二重TaqMan MGB FQ-PCR, 检测

Abstract: To detect the African swine fever virus (ASFV) and Classical swine fever virus (CSFV) wild strains rapidly, a specific and sensitive real-time fluorescence quantitative PCR (FQ-PCR) method was established by using the TaqMan MGB probe technique. Specific primers and probes were designed based on the P72 gene of ASFV and 5' non-coding region of the CSFV wild strain to establish the duplex FQ-PCR assay based on TaqMan MGB probe technique. The sensitivity, specificity and stability of FQ-PCR were determined, and 50 clinical samples were simultaneously detected by the FQ-PCR assay, the national standard of CSFV and the OIE recommended detection method for ASFV. The results indicated that the duplex FQ-PCR assay was successfully established and showed a good linear relationship at a template range of 10⁰-10⁶ copies·μL^-1; the specificity of duplex FQ-PCR assay revealed that amplifications were showed synthetic on ASFV and CSFV genes, but other pathogens have no amplifications; variation coefficient of intra and inter assay were 1.18%-2.08%, respectively; the sensitivity of the assay was 10 copies·μL^-1; Fifty clinical samples was detected by the duplex FQ-PCR, the results was consistent with detection of CSFV by national standard and detection of ASFV by OIE recommended method. The duplex TaqMan MGB FQ-PCR assay of ASFV and CSFV wild strain was specific, sensitive, rapid and suitable for identify detection of ASFV and CSFV wild strains.

Key words: African swine fever virus, classical swine fever virus wild strains, duplex TaqMan MGB FQ-PCR, detection

中图分类号:

王淑娟, 班付国, 王东方, 刘影, 赵雪丽, 谢彩华, 王翠, 马震原, 杨海波, 柴茂, 闫若潜. 鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用[J]. 畜牧兽医学报, 2021, 52(1): 177-184.

WANG Shujuan, BAN Fuguo, WANG Dongfang, LIU Ying, ZHAO Xueli, XIE Caihua, WANG Cui, MA Zhenyuan, YANG Haibo, CHAI Mao, YAN Ruoqian. Establishment and Application of a Duplex TaqMan MGB FQ-PCR for Differential Detection of African Swine Fever Virus and Classical Swine Fever Virus Wild Strains[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 177-184.

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