Development of multiplex TaqMan qPCR for simultaneous detection and differentiation of eight common swine viral and bacterial pathogens

Abstract

It is laborious to diagnose the infections of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Suid herpesvirus 1 (SuHV-1) because of the similar clinical symptoms in piglets. Staphylococcus aureus (S. aureus), Streptococcus suis (S. suis), Salmonella choleraesuis (S. choleraesuis, serotype: 6,7:c:1,5), and Escherichia coli (E. coli) are common secondary bacterial pathogens in viral infections. Furthermore, the mixed infection of these viral and bacterial pathogens is more and more common in practical swine breeding. Therefore, a TaqMan multiplex qPCR method for simultaneous detection and differentiation of their pathogen was established in this study by designing specific primers and probes for the E2 gene of CSFV, the ORF7 gene of PRRSV, the ORF1 gene of PCV2 and the gE gene of SuHV-1, the nuc gene of S. aureus, the ef-tu gene of S. suis, the ivnA gene of S. choleraesuis, and the 23S rRNA gene of E. coli, and its specificity, sensitivity, and reproducibility were subsequently tested. The results showed that TaqMan multiplex qPCR method showed a high specificity with no cross reaction between different viruses, and a good repeatability with its coefficient of variation lower than 5%. Besides, the sensitivity of this method was also at least 10 times higher compared with conventional PCR. Overall, this study provided a reliable multiplex TaqMan qPCR method for the diagnosis and differentiation of the mentioned pathogens in pigs, laying a certain technical basis for disease prevention and control.

Keywords: Bacterium; Multiplex TaqMan qPCR; Virus.

Fig. 1

Agarose gel electrophoresis of PCR products. A CSFV 105 bp, PRRSV 133 bp, PCV2 121 bp, SuHV-1 100 bp. B S. aureus 125 bp, S. suis 139 bp, S. choleraesuis 130 bp, E. coli 134 bp. M, DL 2 000 DNA ladder molecular weight marker; NTC, negative control

Fig. 2

The multiplex TaqMan qPCR amplification plot showing specific amplification of positive samples. Specific amplification of A CSFV, PRRSV, PCV2, and SuHV-1, and B S. aureus, S. suis, S. choleraesuis, and E. coli by the multiplex TaqMan qPCR, and no reactivity with A Marc-145, PK-15 cells, PPV, BVDV, PEDV, and TGEV; B P. multocida and C. perfringens; and NTC. NTC, negative control

Fig. 3

Amplification curve and standard curves of the multiplex TaqMan qPCR for CSFV, PRRSV, PCV2, SuHV-1, S. aureus, S. suis, S. choleraesuis, and E. coli, respectively. A Plasmid DNA (10⁷ to10³) of CSFV, PRRSV, PCV2, and SuHV-1 were used for establishing the standard curve by the multiplex TaqMan qPCR. B Plasmid DNA (10⁷ to10³) of S. aureus, S. suis, S. choleraesuis, and E. coli were used for establishing the standard curve by the multiplex TaqMan qPCR

Fig. 4

Sensitivity of the multiplex TaqMan qPCR and conventional PCR. The multiplex TaqMan qPCR was at least 10 times more sensitive than the conventional PCR. A Standard serially diluted plasmid DNA (10⁵ to10⁰) of CSFV, PRRSV, PCV2, and SuHV-1 used for detection limit of the developed assay by the multiplex TaqMan qPCR and conventional PCR, respectively. B Standard serially diluted plasmid DNA (10⁵ to10⁰) of S. aureus, S. suis, S. choleraesuis, and E. coli used for detection limit of the developed assay by the multiplex TaqMan qPCR and conventional PCR, respectively. M, DL 2 000 DNA ladder molecular weight marker. 1–6, 10⁵ to10⁰ Standard serially diluted plasmid DNA. NTC, negative control