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. 2023 Jun 18;10(6):402.
doi: 10.3390/vetsci10060402.

Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China

Jianpeng Chen  1 , Rongchao Liu  1 , Huaicheng Liu  1 , Jing Chen  1 , Xiaohan Li  1 , Jianfeng Zhang  2 , Bin Zhou  1
Affiliations

Development of a Multiplex Quantitative PCR for Detecting Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Deltacoronavirus Simultaneously in China

Jianpeng Chen et al. Vet Sci. .

Abstract

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) belong to the category of swine enteric coronavirus that cause acute diarrhea in piglets, which has resulted in massive losses to the pig husbandry. Therefore, a sensitive and rapid detection method which can differentially detect these viruses that lead to mixed infections in clinical cases, is urgently needed. According to the conserved regions of the PEDV M gene, TGEV S gene, and PDCoV N gene, and the reference gene of porcine (β-Actin), we designed new specific primers and probes for the multiplex qPCR assay capable of simultaneously detecting three RNA viruses. This method, with a great specificity, did not cross-react with the common porcine virus. Moreover, the limit of detection of the method we developed could reach 10 copies/μL ,and the intra- and inter-group coefficients of variation of it below 3%. Applying this assay to detect 462 clinical samples which were collected in 2022-2023, indicated that the discrete positive rates of PEDV, TGEV, and PDCoV were 19.70%, 0.87%, and 10.17%, respectively. The mixed infection rates of PEDV/TGEV, PEDV/PDCoV, TGEV/PDCoV, and PEDV/TGEV/PDCoV were 3.25%, 23.16%, 0.22%, and 11.90%, respectively. All in all, the multiplex qPCR assay we developed as a tool for differential and rapid diagnosing can be put on the active prevention and control of PEDV, TGEV, and PDCoV, , which can create great value in the diagnosis of swine diarrhea diseases.

Keywords: detection; mixed infection; multiplex quantitative PCR; porcine deltacoronavirus; porcine epidemic diarrhea virus; transmissible gastroenteritis virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Standard curves of the multiplex qPCR assay.
Figure 2
Figure 2
Amplified curves of the specificity test of the triplex quantitative PCR method. (A) Specificity test of PEDV; (B): specificity test of TGEV; and (C): specificity test of PDCoV.
Figure 3
Figure 3
The test for the sensitivity of the triplex quantitative PCR method. (A) The test for the sensitivity of PEDV; (B) the test for the sensitivity of TGEV; and (C) the test for the sensitivity of PDCoV.
Figure 3
Figure 3
The test for the sensitivity of the triplex quantitative PCR method. (A) The test for the sensitivity of PEDV; (B) the test for the sensitivity of TGEV; and (C) the test for the sensitivity of PDCoV.
Figure 4
Figure 4
The detailed information of the clinical samples using PCR. (A) PCR product of samples for detecting PEDV; (B) PCR product of samples for detecting TGEV; and (C) PCR product of samples for detecting PDCoV.
Figure 5
Figure 5
Epidemiological distribution maps of PEDV/TGEV/PDCoV in clinical samples from five provinces in China.
See this image and copyright information in PMC

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References

    1. Zhang J., Tsai Y.L., Lee P.Y.A., Chen Q., Zhang Y., Chiang C.J., Shen Y.H., Li F.C., Chang H.F.G., Gauger P.C., et al. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus. J. Virol. Methods. 2016;234:34–42. doi: 10.1016/j.jviromet.2016.03.016. - DOI - PMC - PubMed
    1. Liu Q., Wang H.Y. Porcine enteric coronaviruses: An updated overview of the pathogenesis, prevalence, and diagnosis. Vet. Res. Commun. 2021;45:75–86. doi: 10.1007/s11259-021-09808-0. - DOI - PMC - PubMed
    1. Wang D., Fang L.R., Xiao S.B. Porcine epidemic diarrhea in China. Virus Res. 2016;226:7–13. doi: 10.1016/j.virusres.2016.05.026. - DOI - PMC - PubMed
    1. Yang Y.L., Yu J.Q., Huang Y.W. Swine enteric alphacoronavirus (swine acute diarrhea syndrome coronavirus): An update three years after its discovery. Virus Res. 2020;285:198024. doi: 10.1016/j.virusres.2020.198024. - DOI - PMC - PubMed
    1. Kocherhans R., Bridgen A., Ackermann M., Tobler K. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes. 2001;23:137–144. doi: 10.1023/A:1011831902219. - DOI - PMC - PubMed

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