First Specific Detection of Mammalian Orthoreovirus from Goats Using TaqMan Real-Time RT-PCR Technology

Li Mao; Xia Li; Xuhang Cai; Wenliang Li; Jizong Li; Shanshan Yang; Junjun Zhai; Sizhu Suolang; Bin Li

doi:10.3390/vetsci11040141

First Specific Detection of Mammalian Orthoreovirus from Goats Using TaqMan Real-Time RT-PCR Technology

Vet Sci. 2024 Mar 22;11(4):141. doi: 10.3390/vetsci11040141.

Authors

Li Mao^{1

2

3

4

5

6}, Xia Li^{1

2

3

4

5}, Xuhang Cai^{1

2

3

4

7}, Wenliang Li^{1

2

3

4

5

6}, Jizong Li^{1

2

3

4

5

6}, Shanshan Yang^{1

2

3

4

6}, Junjun Zhai⁸, Sizhu Suolang⁵, Bin Li^{1

2

3

4

5

6}

Affiliations

¹ Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
² Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014, China.
³ National Center for Engineering Research of Veterinary Bio-Products, Nanjing 210014, China.
⁴ Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China.
⁵ College of Animal Science, Tibet Agricultural and Animal Husbandry University, Linzhi 860000, China.
⁶ Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225000, China.
⁷ College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China.
⁸ Shaanxi Province Engineering and Technology Research Center of Cashmere Goat, Yulin University, Yulin 719000, China.

Abstract

Mammalian orthoreovirus (MRV) infections are ubiquitous in multiple mammalian species including humans, and mainly causes gastroenteritis and respiratory disease. In this study, we developed a rapid and sensitive TaqMan qRT-PCR method for MRV detection based on the primers and probe designed within the conserved L1 gene. The qRT-PCR assay was evaluated for its sensitivity, specificity, efficiency and reproducibility. It was found that the detection sensitivity was equivalent to 10 DNA copies/μL, and the standard curves had a linear correlation of R² = 0.998 with an amplification efficiency of 99.6%. The inter- and intra-assay coefficients of variation (CV%) were in the range of 0.29% to 2.16% and 1.60% to 3.60%, respectively. The primer sets specifically amplified their respective MRV segments and had the highest detection sensitivities of 10^0.25 TCID₅₀/mL with amplification efficiencies of 99.5% (R² = 0.999). qRT-PCR was used for MRV detection from samples of sheep, goats, and calves from four regions in China, and the overall MRV prevalence was 8.2% (35/429), whereas 17/429 (4.0%) were detected by RT-PCR and 14/429 (3.3%) by virus isolation. The qRT-PCR assay showed significantly higher sensitivity than RT-PCR and virus isolation. Results from an epidemiological survey indicated that the positive rate of MRV in rectal swabs from sheep and goats tested in Shaanxi, Jiangsu, and Xinjiang were 9/80 (11.3%), 12/93 (12.9%) and 14/128 (10.9%), respectively. In goats and sheep, MRV prevalence was obviously associated with season and age, with a high positive rate of more than 8% during September to April and approximately 13% in small ruminant animals under two months of age. This is the first instance of MRV infection in sheep and goats in China, thus broadening our knowledge of MRV hosts. Consequently, primer optimization for qRT-PCR should not only prioritize amplification efficiency and specificity, but also sensitivity. This assay will contribute to more accurate and rapid MRV monitoring by epidemiological investigation, viral load, and vaccination efficacy.

Keywords: TaqMan qRT-PCR; detection; goat; mammalian orthoreovirus; sheep.

Abstract

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