Development of a Multiplex RT-PCR Assay for Simultaneous Detection of Four Potential Zoonotic Swine RNA Viruses

Figures

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  Figure 1. mRT-PCR optimization and assay specificity. M: 2000 bp marker, 1: SaV, 2: EMCV, 3: RVA and 4: AstV, P: standard plasmid template mix containing SaV, EMCV, RVA and AstV fragments. (A) Optimization of mRT-PCR using plasmids containing SaV, EMCV, RVA and AstV fragments. (B) Assay specificity. N: negative control, 1: SaV, 2: EMCV, 3: RVA, 4: AstV and 5–10: PEDV, SVV, TGEV, PRRSV, PDCoV and PSV, respectively.

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  Figure 2. mRT-PCR assay sensitivity. The sensitivity of mRT-PCR and sRT-PCR assays was checked using 10-fold serially diluted plasmids containing SaV, EMCV, RVA and AstV fragments as a template. M: DL2000 DNA Marker, 1: 2.5 × 106 copies, 2: 2.5 × 105 copies, 3: 2.5 × 104 copies, 4: 2.5 × 103 copies, 5: 2.5 × 102 copies, 6: 2.5 × 101 copies and 7: 2.5 × 100 copies. (A) mRT-PCR, (B) SaV, (C) EMCV, (D) RVA, and (E) AstV.

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  Figure 3. The number of single and mixed infections of Ast, EMCV, RVA and SaV. The coinfection rate of each virus was calculated using 100 samples that tested positive for at least one virus.

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  Figure 4. Phylogenetic analysis of SaV, EMCV, RVA and AstV. The strains identified in this study (marked with red dots) and reference strains from GenBank were used to construct phylogenetic trees. Scales indicate units of the number of base substitutions per site. (A) Phylogenetic tree of SaV. The tree was built using 224 positions from the ORF1 region of SaV. (B) Phylogenetic tree of EMCV. The tree was constructed using 211 positions from the 3D region of the EMCV. Mengovirus was used as an outgroup. (C) Phylogenetic tree of RVA. The tree was built based on 545 positions of the VP6 region. Rotavirus A from dove (avian) was used as an outgroup. (D) Phylogenetic tree of AstV. The tree was built based on 379 positions of the ORF1ab region. As an outgroup, Turkey (avian) Astrovirus is included.