A SYBR-Green <b>Ⅰ</b> RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats

ZHANG Jingpeng; JIANG Jinxiu; LIN YuSheng; YOU Wei; LIU Daoquan; MAO Kunming; JIANG Bin; Hu Qilin

doi:10.19303/j.issn.1008-0384.2021.07.007

Volume 36 Issue 7

Jul. 2021

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Article Navigation > Fujian Journal of Agricultural Sciences > 2021 > 36(7): 779-784

ZHANG J P, JIANG J X, LIN Y S, et al. A SYBR-Green Ⅰ RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats [J]. Fujian Journal of Agricultural Sciences，2021，36（7）：779−784 doi: 10.19303/j.issn.1008-0384.2021.07.007

Citation:

ZHANG J P, JIANG J X, LIN Y S, et al. A SYBR-Green Ⅰ RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats [J]. Fujian Journal of Agricultural Sciences，2021，36（7）：779−784 doi: 10.19303/j.issn.1008-0384.2021.07.007

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A SYBR-Green Ⅰ RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats

doi: 10.19303/j.issn.1008-0384.2021.07.007

1.
Institute of Animal Husbandry and Veterinary Medicine, Fuzhou, Fujian　350013, China
2.
Fuzhou Animal Disease Prevention and Control Center, Fuzhou, Fujian　350002, China
3.
Fuqing Technical Service Center of Animal Husbandry and Veterinary Medicine, Fuzhou, Fujian　350300, China

Received Date: 2020-12-04
Rev Recd Date: 2021-03-25

Available Online: 2021-07-13

Publish Date: 2021-07-28

Abstract

Abstract

Objective A rapid, sensitive detection method for early diagnosis and epidemiological survey on enzootic nasal tumor (ENT) in goats was established. Method Bioinformatics methods were employed for sequence alignment of ENTV-2 with ENTV-1, ERVs, and JSRV in search for the conserved sequence of the virus. Primers for qPCR were designed to establish a SYBR-Green I RT-qPCR methodology for its detection. Reaction conditions were optimized, and a standard positive plasmid used to determine the specificity, sensitivity, and reproducibility of the newly developed assay. Result A linear standard curve was found for the detection with a R²=0.992. The method specifically detected only ENTV-2, not the highly homologous ERVs, nor amplified ORFV, MO, or Mmc. It showed a detection sensitivity of up to 7.51×10² copies·μL⁻¹, which was 100 times greater than the conventional PCR can deliver, a repeatability with a coefficient variations (CV) of less than 1% on intra- and inter-batch tests, and a positive detection rate of 17.3% on 81 clinical samples. Conclusion The newly established SYBR-Green I RT-qPCR was specific, sensitive, repeatable, and considered adequate for early and rapid detection of ENTV-2 in goats.
- Enzootic nasal tumor virus of goats(ENTV-2),
- SYBR-Green Ⅰ,
- RT-qPCR

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References(20)

References

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