Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus

Xinyou Yu, Tong Li, Tianzhi Li, Lin Dong, Jinliang Wang, Zhiqiang Shen

2022

Abstract

The present study envisaged the development of a fluorescence PCR test for the simultaneous detection of the African swine fever virus (ASFV) and the porcine epidemic diarrhea virus (PEDV) by designing specific primers based on the sequences of the p72 gene of ASFV and the N gene of PEDV in GenBank. Subsequently, the sample loading system and the PCR program was optimized to establish a dual SYBR Green I fluorescence PCR assay for ASFV and PEDV. Furthermore, the specificity, sensitivity and repeatability of the established assay were evaluated. Finally, the established PCR assay was tested using clinical samples. The results demonstrated that the optimal loading amount of each primer in a 20 µL reaction system was: F1 0.5 µL, R1 0.8 µL, F2 1.5 µL and R2 1.2 µL; the optimal PCR program was: reverse transcription at 42 ℃ for 5 min; pre-denaturation at 95°C for 2 min; and 40 cycles of (denaturation at 94°C for 5 s followed by annealing at 53°C for 25 s, where the fluorescence was collected). The established assay exhibited a good specificity and did not cross-react with other common swine viruses. The sensitivity of detecting the ASFV and PEDV was 8.8 copies/µL and 3.7 copies/µL, respectively, and the within-run and between-run coefficients of variation of Tm values were not more than 1.0%. The test results of 162 clinical samples using the established PCR assay were consistent with the reference methods. The dual SYBR Green I fluorescence PCR assay established in this study for detecting ASFV and PEDV showed high sensitivity and good specificity, and it can be used for the rapid detection of these two clinical diseases.

Paper Citation

in Harvard Style

Yu X., Li T., Li T., Dong L., Wang J. and Shen Z. (2022). Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus. In Proceedings of the 8th International Conference on Agricultural and Biological Sciences - Volume 1: ABS, ISBN 978-989-758-607-1, pages 5-10. DOI: 10.5220/0011594000003430

in Bibtex Style

@conference{abs22,
author={Xinyou Yu and Tong Li and Tianzhi Li and Lin Dong and Jinliang Wang and Zhiqiang Shen},
title={Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus},
booktitle={Proceedings of the 8th International Conference on Agricultural and Biological Sciences - Volume 1: ABS,},
year={2022},
pages={5-10},
publisher={SciTePress},
organization={INSTICC},
doi={10.5220/0011594000003430},
isbn={978-989-758-607-1},
}

in EndNote Style

TY - CONF

JO - Proceedings of the 8th International Conference on Agricultural and Biological Sciences - Volume 1: ABS,
TI - Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus
SN - 978-989-758-607-1
AU - Yu X.
AU - Li T.
AU - Li T.
AU - Dong L.
AU - Wang J.
AU - Shen Z.
PY - 2022
SP - 5
EP - 10
DO - 10.5220/0011594000003430