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. 2019 Jun;103(12):4943-4952.
doi: 10.1007/s00253-019-09835-7. Epub 2019 Apr 26.

A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

Xin Huang  1   2 , Jianing Chen  2 , Gang Yao  1 , Qingyong Guo  1 , Jinquan Wang  3 , Guangliang Liu  4   5
Affiliations

A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

Xin Huang et al. Appl Microbiol Biotechnol. 2019 Jun.

Abstract

Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R2) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.

Keywords: Diagnosis; Multiplex real-time RT-qPCR; Swine enteric coronaviruses; TaqMan probe.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Establishment of single real-time RT-qPCR assay for the individual virus. a The amplification curves (top) and a standard curve (bottom) for detection of PEDV M gene were generated. The probe was labeled with FAM at 5′-end and TAMRA at 3′-end. b The amplification curves (top) and a standard curve (bottom) for detection of PDCoV M gene were generated. The probe was labeled with Cy5 at 5′-end and BHQ2 at 3′-end. c The amplification curves (top) and a standard curve (bottom) for detection of TGEV N gene were generated. The probe was labeled with HEX at 5′-end and BHQ1 at 3′-end. d The amplification curves (top) and a standard curve (bottom) for detection of PEDV N gene were generated. The probe was labeled with TexasRed at 5′-end and BHQ2 at 3′-end
Fig. 2
Fig. 2
Establishment of multiplex real-time RT-qPCR. a Amplification curves and b standard curves of optimized multiplex TaqMan-probe-based real-time RT-qPCR for detection of PEDV, PDCoV, TGEV, and PEAV were generated at the optimum amplification conditions. The correlation coefficient and amplification efficacy of the standards curves, PEDV (R2 = 0.999; Eff% = 103.5), PDCoV (R2 = 0.999; Eff% = 104.7), TGEV (R2 = 0.999; Eff% = 105.6), and PEAV (R2 = 1; Eff% = 100.4), were ideal for detecting the target genes
Fig. 3
Fig. 3
The specificity of multiplex TaqMan-probe-based real-time RT-qPCR. 1–4: positive templates of TGEV, PDCoV, PEDV, and PEAV. 5–14: templates of PAsV, PKV, FMDV-O, FMDV-A, CSFV, PRRSV, PRV, PCV2, RrV, and nuclease-free water control
Fig. 4
Fig. 4
Co-infection analysis between detected swine enteric coronaviruses. A total of 354 clinical diarrhea samples collected from six provinces of China between 2015 and 2018 were subjected to an analysis by the real-time RT-qPCR assay developed in this study. The Venn diagram shows the number of samples infected by either single virus or multi-coronaviruses
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