针对非洲猪瘟病毒MGF360-13L基因的TaqMan荧光定量PCR的建立

doi:10.3864/j.issn.0578-1752.2021.05.018

摘要/Abstract

摘要：

【目的】建立一种以MGF360-13L基因为靶标的实时荧光定量PCR检测非洲猪瘟病毒（African swine fever virus, ASFV）的方法,为非洲猪瘟（African swine fever, ASF）的诊断、MGF360-13L基因缺失毒株鉴别、病毒分离鉴定、基因功能研究提供技术支持。【方法】首先,以ASFV中国流行毒株（GenBank: MK333180.1）的MGF360-13L基因序列为靶标设计并筛选了1对特异性引物和探针,建立其荧光定量PCR检测方法。设计引物13L-F/13L-R,扩增MGF360-13L,并将其克隆至pOK12载体,挑取阳性克隆并测序验证,构建重组质粒标准品。将重组质粒标准品连续10倍梯度稀释,以稀释后的各梯度质粒标准品为模板,配制反应体系和设置反应条件后进行荧光定量PCR检测,建立标准曲线,并对其敏感性和重复性进行评价;其次,以连续10倍梯度稀释的重组质粒标准品为模板,利用引物13L-F/13L-R进行常规PCR检测,以比较荧光定量PCR的敏感性。最后,运用本检测方法和本研究组已建立的针对ASFV p72的荧光定量PCR检测方法同时对黑龙江某猪场发生ASF时采集的30份临床样品进行检测,以比较两种检测方法的符合率。另外,应用该方法对感染原代巨噬细胞的ASFV野毒株和MGF基因缺失毒株进行鉴别检测。【结果】利用引物13L-F/13L-R可扩增出一条800 bp左右的特异性目的条带,而阴性对照无条带,成功构建出标准品。本研究建立的实时荧光定量PCR检测方法在质粒标准品为1.56×10¹-1.56×10⁸拷贝/μL时,呈现出良好的线性关系,线性回归方程为：y =-3.295 lg(x) + 45.995,线性相关系数R²为0.997,对ASFV核酸最低检测限为15.6拷贝/μL。在特异性检验中,除ASFV核酸外,猪瘟病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪圆环病毒1型、猪圆环病毒2型等病毒核酸均未出现扩增曲线,表明该方法的特异性良好。与最低检测限为1.56×10⁴拷贝/μL病毒核酸的常规PCR检测方法相比,本研究建立的TaqMan荧光定量PCR检测方法高出其约3个数量级,具有较高的敏感性。在临床样品检测中,两种检测方法经过McNemar检验,P = 0.5>0.05,表明两种检测方法的结果无统计学差异;经过Kappa检验,Kappa = 0.867>0.75,P<0.001,提示两种检测方法符合率较好,能够有效区分ASFV野毒株和MGF360-13L基因缺失毒株。【结论】建立的TaqMan荧光定量PCR特异、敏感、重复性好,不仅丰富了ASFV现有检测靶标,也为后续MGF360-13L功能、MGF缺失毒株的鉴定及相应基因缺失疫苗株的鉴别诊断提供技术支持。

关键词: 非洲猪瘟病毒, MGF360-13L, 荧光定量PCR, 鉴别诊断

Abstract:

【Objective】The objective of this study is to develop a TaqMan real-time PCR for detection of African swine fever virus (ASFV) MGF360-13L gene, providing technical support for diagnosis, virus purification, differential diagnosis of MGF360-13L deletion ASFV strains, and gene function research of ASFV.【Method】In this study, the TaqMan real-time PCR primers and probes were designed based on the ASFV MGF360-13L (GenBank: MK333180.1), and the TaqMan real-time quantitative PCR assay of ASFV was established. A pair of specific primers, 13L-F/13L-R was designed to construct ASFV standard plasmid. The standard curve was generated for quantitative analysis using the ten-fold dilution of the ASFV standard plasmid, and the sensitivity and repeatability of the system were also evaluated. The ten-fold dilution of the ASFV standard was also detected by PCR to determine the sensitivity of the method. Thirty clinical samples collected from an outbreak ASF pig farm in Heilongjiang province were simultaneously tested by this TaqMan real-time PCR, and another one previously established in our laboratory to compare the coincidence rate of these two detection methods. Differential diagnosis of parent ASFV and MGF gene deletion strain after infection with PAM cell【Result】A specific band of about 800 bp was amplified using primer 13L-F/13L-R, and no band was found in the negative control, and the standard plasmid was successfully constructed. The standard curve had a good linear relationship between real-time PCR cycle threshold (Ct) and template copies. The linear regression equation was: y = -3.295 lg(x) + 45.995 with correlation coefficient of 0.997, and the minimum detection limit was 15.6 copies/μL of ASFV nucleic acid. There was no cross-reaction with classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, porcine circovirus type 1, and porcine circovirus type 2. In the clinical sample test, the results of the two methods in McNemar test, P = 0.5>0.05, indicating that there was no statistical difference between the two methods and in Kappa test, Kappa = 0.867>0.75, P<0.001, suggesting it was a good agreement and could effectively identify parent ASFV or MGF gene deletion ASFV infection. 【Conclusion】The TaqMan real-time PCR for detection of ASFV MGF360-13L established in this study had highly specific, sensitive, reproducible, and high coincidence rate. It not only enriched the detection method of ASFV, but also provided technical support for researching the gene function of MGF360-13L, identification of the MGF360-13L gene-deleted ASFV, and differential diagnosis of related gene deletion vaccine strains.

Key words: African swine fever virus, MGF360-13L, TaqMan real-time PCR, detection

王涛,韩玉,潘力,王冰,孙茂文,王翌,罗玉子,仇华吉,孙元. 针对非洲猪瘟病毒MGF360-13L基因的TaqMan荧光定量PCR的建立[J]. 中国农业科学, 2021, 54(5): 1073-1080.

Tao WANG,Yu HAN,Li PAN,Bing WANG,MaoWen SUN,Yi WANG,YuZi LUO,HuaJi QIU,Yuan SUN. Development of a TaqMan Real-Time PCR Targeting the MGF360-13L Gene of African Swine Fever Virus[J]. Scientia Agricultura Sinica, 2021, 54(5): 1073-1080.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.05.018

https://www.chinaagrisci.com/CN/Y2021/V54/I5/1073

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标准品（拷贝） Standard plasmid template (copies/μL)	组内重复性 Intra-assay		组间重复性 Inter-assay
标准品（拷贝） Standard plasmid template (copies/μL)	平均值Mean ± SD	变异系数 CV(%)	平均值 Mean ± SD	变异系数CV(%)
1.56 × 10⁹	16.33 ± 0.09	0.54	16.23 ± 0.26	1.63
1.56 × 10⁸	19.96 ± 0.09	0.28	20.0 ± 0.12	0.6
1.56 × 10⁷	22.89 ± 0.08	0.35	22.56 ± 0.23	1.0
1.56 × 10⁶	26.13 ± 0.08	0.85	25.76 ± 0.5	1.93