采用双重 SYBR Green I 实时 PCR 法对临床样本中鹅细小病毒和星状病毒的同时鉴别诊断。
Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.
机构信息
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Shushan District West Yangtze River Road 130#, Hefei, 230036, Anhui Province, People's Republic of China.
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Shushan District West Yangtze River Road 130#, Hefei, 230036, Anhui Province, People's Republic of China.
出版信息
Mol Cell Probes. 2020 Aug;52:101561. doi: 10.1016/j.mcp.2020.101561. Epub 2020 Mar 12.
Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 10 and 6.58 × 10 copies/μL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.
设计了两对引物，用于结合鹅细小病毒（GPV）和鹅星状病毒（GAstV）的保守基因组区域，建立了一种简单、敏感、高度特异的双重实时定量 PCR（qPCR）方法，可同时检测这两种病毒。通过各自熔解曲线的峰，该双重 qPCR 可以区分 GPV（熔点：82.1°C）和 GAstV（熔点：79.8°C）。与其他水禽病毒的混合测试未产生非特异性峰。建立的标准曲线显示出良好的线性关系（R>0.997），GPV 和 GAstV 的检测限（LOD）分别为 5.74×10 和 6.58×10 拷贝/μL。内和间试验的变异系数均<2%，表明该方法具有良好的重复性。用双重 qPCR 检测和常规 PCR 检测了 20 份患病鹅的组织样本。双重 qPCR 检测到 GPV 的阳性率为 25%，GAstV 的阳性率为 45%，GPV 和 GAstV 混合感染的阳性率为 15%，略高于常规 PCR 的结果。这些结果表明，该双重 qPCR 方法高度敏感、特异、可重复，适用于流行病学研究，以有效控制 GPV 和 GAstV 的传播。
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