Development of a Quadruplex RT-qPCR for the Detection of Porcine Astrovirus, Porcine Sapovirus, Porcine Norovirus, and Porcine Rotavirus A

Abstract

Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09-1.24% for intra-assay and 0.08-1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases.

Keywords: multiplex RT-qPCR; porcine astrovirus; porcine norovirus; porcine rotavirus species A; porcine sapovirus.

Figure 1

The amplification curves of p-PoAstV (A), p-PoSaV (B), p-PoNoV (C), and p-PoRVA (D), and the standard curves (E). In (A–D), 1–7: the concentrations of standard plasmid constructs ranged from ${10}^7$ to ${10}^1$ copies/µL; 8: nuclease-free distilled water. In (E), 1–4: the standard curves of p-PoAstV, p-PoSaV, p-PoNoV, and p-PoRVA, respectively.

Figure 2

Specificity assessment of the quadruplex RT-qPCR. 1: p-PoAstV; 2: p-PoRVA; 3: p-PoNoV; 4: p-PoSAV; 5–8: the positive clinical samples of PoAstV, PoRVA, PoNoV, and PoSaV; 9: PoAstV (astrovirus 3 strain); 10: PoRVA (G5 strain); 11: PoNoV (GII.11 strain); 12: PoSaV (G3 strain); 13–22: ASFV, FMDV, CSFV, PRRSV, PRV, SIV, PEDV, TGEV, PDCoV, and PCV2; 23: negative fecal sample; 24: distilled water.

Figure 3

Sensitivity assessment of the quadruplex RT-qPCR. In (A–D), 1–9: the concentrations of standard plasmid constructs ranged from ${10}^7$ to ${10}^{-1}$ copies/μL (final reaction concentrations); 10: negative control.

Figure 4

Assessment of sensitivity using probit regression analysis. The p-PoAstV (A), p-PoSaV (B), p-PoNoV (C), and p-PoRVA (D) had LODs of 138.001 (95% CI of 126.235–160.439), 135.167 (95% CI of 123.470–156.146), 140.732 (95% CI of 128.740–165.327), and 132.199 (95% CI of 120.391–152.436), respectively.