用于非洲猪瘟病毒野生型毒株以及缺失MGF505-2R、EP402R和I177L基因毒株差异检测的多重实时定量PCR检测方法的建立
The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus.
作者信息
Zhao Kang, Shi Kaichuang, Zhou Qingan, Xiong Chenyong, Mo Shenglan, Zhou Hongjin, Long Feng, Wei Haina, Hu Liping, Mo Meilan
机构信息
College of Animal Science and Technology, Guangxi University, Nanning 530005, China.
Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
出版信息
Animals (Basel). 2022 Jul 8;12(14):1754. doi: 10.3390/ani12141754.
African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/μL for the B646L (p72) gene, and 3.21 copies/μL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.
非洲猪瘟病毒（ASFV）可引发非洲猪瘟（ASF），这是一种在家猪和野猪中极具毁灭性的出血性疾病。目前，已证实缺失MGF505R、EP402R（CD2v）和I177L基因的ASFV毒株是理想的疫苗候选毒株。为开发一种区分野生型和基因缺失型ASFV毒株的检测方法，设计了四对靶向ASFV B646L（p72）、I177L、MGF505 - 2R和EP402R（CD2v）基因的特异性引物和TaqMan探针。在优化反应条件（包括退火温度、引物浓度和探针浓度）后，开发了一种用于差异检测野生型和基因缺失型ASFV毒株的多重实时荧光定量PCR检测方法。结果表明，该多重实时荧光定量PCR检测方法能够特异性检测ASFV B646L（p72）、I177L、MGF505 - 2R和EP402R（CD2v）基因，其中B646L（p72）基因的检测限（LOD）为32.1拷贝/μL，I177L、MGF505 - 2R和EP402R（CD2v）基因的检测限为3.21拷贝/μL。然而，该检测方法无法检测经典猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪流行性腹泻病毒（PEDV）、猪三角洲冠状病毒（PDCoV）、猪圆环病毒2型（PCV2）、PCV3和伪狂犬病病毒（PRV）。该检测方法具有良好的重复性和再现性，批内和批间变异系数（CV）均小于1.56%。该检测方法用于检测4239份临床样本，结果显示12.60%（534/4239）的样本ASFV呈阳性，其中10份样本缺失EP402R基因，6份样本缺失MGF505 - 2R基因，14份样本同时缺失EP402R和MGF505 - 2R基因。结果表明，本研究开发的多重实时荧光定量PCR可为ASFV B646L、I177L、MGF505 - 2R和EP402R基因的差异检测提供一种快速、灵敏且特异的诊断工具。
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