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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

作者： Xu, Zhuoran 1 ; Liu, Hongwei 1 ; Zheng, Xin 2 ; Cheng, Xiaoxia 1 ; Wang, Shao 1 ; You, Guangju 1 ; Zhu, Xiaoli 1 ; Zheng, Min 1 ; Dong, Hui 1 ; Xiao, Shifeng 1 ; Zeng, Li 1 ; Zeng, Xiancheng 2 ; Chen, Shaoying 1 ; Chen, Shilong 1 ;

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

2.Fujian Agr & Forestry Univ, Coll Anim Sci, Fuzhou, Peoples R China

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

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 ）

ISSN：

年卷期： 2024 年 11 卷

页码：

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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