Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR
Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR
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DOI：
10.1016/j.mcp.2020.101561
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发表时间：
2020-08-01
影响因子：
3.3
通讯作者：
Qi, Ke-zong
中科院分区：
文献类型：
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作者：
Yang, Kan-kan;Xu, Liang;Qi, Ke-zong
针对鹅细小病毒（GPV）和鹅星状病毒（GAstV）基因组保守区域设计2对引物，建立一种简便、灵敏、特异的双重定量PCR（qPCR）方法，用于同时检测GPV和GAstV。双链qPCR可以通过GPV（熔点：82.1 ° C）和GAstV（熔点：79.8 ° C）各自的解链曲线的峰来区分它们。与其他水禽病毒混合检测未产生非特异性峰。GPV和GAstV的标准曲线线性关系良好（R-2 > 0.997），检测限分别为5.74 × 10 ~（-1）和6.58 × 10 ~（-1）拷贝/μ L。批内和批间变异系数均<2%，表明该方法具有良好的重复性。采用双重qPCR和常规PCR检测了20份病鹅组织样品。双重qPCR检测GPV和GAstV的阳性率分别为25%和45%，GPV和GAstV混合感染的阳性率为15%，略高于常规PCR检测结果。该方法灵敏度高、特异性强、重复性好，适用于GPV和GAStV的流行病学研究，可有效控制GPV和GAStV的传播。
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 degrees C) and GAstV (melting point: 79.8 degrees 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-2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 x 10(1) and 6.58 x 10(1) copies/mu L, 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.