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A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus

文献类型： 外文期刊

作者： Zheng, Min 1 ; Chen, Xiuqin 1 ; Wang, Shao 1 ; Wang, Jingxiang 1 ; Huang, Meiqing 1 ; Xiao, Shifeng 1 ; Cheng, Xiaoxia 1 ;

作者机构： 1.Fujian Acad Agr Sci, Inst Anim Husb & Vet Med, Fuzhou 350013, Peoples R China

2.Fujian Anim Dis Control Technol Dev Ctr, Fuzhou 350013, Peoples R China

3.Agr & Rural Bur, Sanming 365000, Peoples R China

关键词： Muscovy duck reovirus; Real-time RT-PCR; TaqMan-MGB probe; Diagnosis; Internal positive control

期刊名称：MOLECULAR AND CELLULAR PROBES （ 影响因子：2.365； 五年影响因子：2.386 ）

ISSN： 0890-8508

年卷期： 2020 年 52 卷

页码：

收录情况： SCI

摘要： 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 x 10(1) copies/mu L 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 beta-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 RTPCR 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 TaqManMGB 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.

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