Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, 350013, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou, 350013, China.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, 350013, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou, 350013, China.
Mol Cell Probes. 2020 Aug;52:101575. doi: 10.1016/j.mcp.2020.101575. Epub 2020 Apr 17.
A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 10 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay. The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection.
描述了一种用于检测临床样本中麝香鸭呼肠孤病毒（Muscovy duck reovirus，MDRV）RNA 的实时逆转录聚合酶链反应（RT-PCR）。该检测基于 TaqMan-MGB 技术，由两条引物和一条探针组成，探针用报告染料 6-羧基荧光素标记，可选择性地结合到 MDRV 的 sigma B 蛋白基因上。该技术还包括一个内部阳性对照（IPC）。实时 RT-PCR 检测能够检测到 MDRV，而其他常见的水禽源性病毒病原体则不能被建立的寡核苷酸组识别，因此表明该检测方法具有特异性。该检测方法的灵敏度为 2.83×10 拷贝/μL，比常规 RT-PCR 高 100 倍。该检测方法的内和间试验变异系数均小于 1.5%，表明该检测方法具有足够的重复性。采用 β-肌动蛋白 mRNA 作为 IPC 以不降低检测效率。该检测方法用于检测 100 个临床样本，结果显示建立的 TaqMan-MGB 实时 RT-PCR 方法的阳性率为 87%（87/100），而常规 RT-PCR 的阳性率为 83%（83/100），两者的符合率为 97.14%。与常规 RT-PCR 相比，TaqMan 荧光定量 RT-PCR 对临床样本的敏感性和阳性率更高。TaqMan-MGB 实时 RT-PCR 检测方法具有高特异性、敏感性和快速性，并且使用 IPC 监测假阴性结果，可使该方法适用于 MDRV 感染的病原监测和流行病学调查。
