Simultaneous detection and differentiation of classical Muscovy duck reovirus and goose-origin Muscovy duck reovirus by RT-qPCR assay with high-resolution melting analysis

文献类型： 外文期刊

第一作者： Xu, Zhuoran

作者： Xu, Zhuoran;Liu, Hongwei;Cheng, Xiaoxia;Wang, Shao;You, Guangju;Zhu, Xiaoli;Zheng, Min;Dong, Hui;Xiao, Shifeng;Zeng, Li;Chen, Shaoying;Chen, Shilong;Xu, Zhuoran;Liu, Hongwei;Zheng, Xin;Zeng, Xiancheng;Cheng, Xiaoxia;Wang, Shao;You, Guangju;Zhu, Xiaoli;Zheng, Min;Dong, Hui;Xiao, Shifeng;Zeng, Li;Chen, Shaoying;Chen, Shilong

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关键词： classical Muscovy duck reovirus; goose-origin Muscovy duck reovirus; duplex real-time RT-PCR; high-resolution melting; differential diagnosis

期刊名称：FRONTIERS IN VETERINARY SCIENCE （ 影响因子：2.9； 五年影响因子：3.3 ）

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年卷期： 2024 年 11 卷

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收录情况： SCI

摘要： Introduction Classical Muscovy duck reovirus (C-MDRV) and goose-origin Muscovy duck reovirus (Go-MDRV) infections cause "Liver white-spots disease" in Muscovy duckling and gosling. It is difficult to differentiate the infections caused by C-MDRV and Go-MDRV using conventional serological methods.Methods Specific primers were designed and synthesized according to sigma NS and lambda A nucleotide sequences of C-MDRV and Go-MDRV, respectively. The PCR amplified products were cloned into the pMD-18-T vector. The recombinant plasmid DNA was used to establish an SYBR Green & Iukcy; based duplex real-time PCR assay for the simultaneous detection and differentiation of C-MDRV and Go-MDRV using high-resolution melting (HRM) analysis. The specificity, sensitivity, and repeatability of the methodology were examined based on the optimization of the reaction system and amplification conditions.Results C-MDRV and Go-MDRV were identified by their distinctive melting temperatures with 84.50 +/- 0.25 degrees C for C-MDRV and 87.50 +/- 0.20 degrees C for Go-MDRV, respectively. The amplifications were specific, and other non-targeted waterfowl viruses employed in this study did not show normalized melting peaks. The intra- and inter-assay coefficients of variations were between 0.05 and 1.83%, demonstrating good repeatability. The detection limits of this assay were 51.4 copiesmu l-1 for C-MDRV and 61.8 copiesmu l-1 for Go-MDRV, respectively. A total of 45 clinical samples were tested by RT-qPCR, with positive rates of 15.56% for C-MDRV and 22.22% for Go-MDRV, without co-infections.Discussion These results suggest that this duplex RT-qPCR method is highly sensitive, specific, and reproducible. The HRM assay established in this study provides a powerful tool for the differential detection and epidemiological investigation of C-MDRV and Go-MDRV.

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