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ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Experimental and Diagnostic Pathology
Volume 11 - 2024 | doi: 10.3389/fvets.2024.1459898

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

Provisionally accepted
Zhuoran Xu Zhuoran Xu 1,2Hongwei Liu Hongwei Liu 1,2Xin Zheng Xin Zheng 2Xiaoxia Cheng Xiaoxia Cheng 1Shao Wang Shao Wang 1Guangju You Guangju You 1Xiaoli Zhu Xiaoli Zhu 3Min Zheng Min Zheng 1Hui Dong Hui Dong 1Shifeng Xiao Shifeng Xiao 1Li Zeng Li Zeng 1Xiancheng Zeng Xiancheng Zeng 2Shaoying Chen Shaoying Chen 1Shilong Chen Shilong Chen 1*
  • 1 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, China
  • 2 College of Animal Science, Fujian agriculture and Forestry University, Fuzhou, Fujian Province, China
  • 3 Fujian Academy of Agricultural Sciences, Fuzhou, China

The final, formatted version of the article will be published soon.

    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 σNS and λA 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 І 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℃ for C-MDRV and 87.50±0.20℃ 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 copies•μL -1 for C-MDRV and 61.8 copies•μL -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.

    Keywords: Classical Muscovy duck reovirus, goose-origin Muscovy duck reovirus, Duplex real-time RT-PCR, high-resolution melting (HRM), differential diagnosis

    Received: 05 Jul 2024; Accepted: 14 Oct 2024.

    Copyright: © 2024 Xu, Liu, Zheng, Cheng, Wang, You, Zhu, Zheng, Dong, Xiao, Zeng, Zeng, Chen and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Shilong Chen, Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, China

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