ABSTRACT
Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.
ABSTRACT
Recently, two novel subgroups of porcine epidemic diarrhea virus (PEDV) were identified in Tibetan pigs on the Qinghai-Tibet Plateau. We further investigated if the novel variant PEDV exists or had been prevalent in Sichuan region. One hundred and sixteen fecal and intestinal tissue samples collected in 2018-2019 from diarrheal pigs of Sichuan were detected for PEDV by RT-PCR, and the molecular characteristics of Spike genes (S) of PEDV were further investigated in this study. The results showed that the detection rate of PEDV was 42.2% (49/116, 95%CI=33.1%-51.8%) in diarrhea samples. Thirteen complete S gene sequences were obtained, and they were 4 149-4 170 bp in length, sharing 94.2%-99.9% identities with each other. Interestingly, SWUN-H3-CH-SCYA-2019 shared 97.0%-98.6% nucleotide sequence identities with those of the novel G1 subgroup of Tibetan Pig PEDVs. Phylogenetic and evolutionary analysis showed that the 13 S genes obtained in this study could be divided into G1 and G2 subgroups, one of which (SWUN-H3-CH-SCYA-2019 strain) fell into the novel G1 subgroup of Tibetan Pig PEDVs;the SWUN-19-CH-SCZY-2018, SWUN-4-CH-SCXC-2018, SWUN-1-CH-SCNJ-2019 and SWUN-3CH-CH-SCZG-2019 were clustered into an unique branch of G2 subgroup, and shared high sequence identities with the novel G2 subgroup of Tibetan Pig PEDVs. To further study the evolution process of 13 PEDVs, the BEAST software was used to estimate the divergence time. The results showed that the divergence time of SWUN-H3-CH-SCYA-2019 was about 2012.3 year, earlier than the earliest divergence time of the other novel G1 subgroup strains (2015.7 year), and the divergence time of SWUN-4-CH-SCXC-2018, SWUN-19-CH-SCZY-2018 and SWUN-3CH-CH-SCZG-2019 were about 2014.2 year, earlier than that of the novel G2 subgroup (2014.7 year). The divergence time of Tibetan pig PEDVs was later than that of strains identified in this study. Tibetan Pig PEDVs were first detected in Sichuan region, and we deduced a conclusion from different divergence time that the novel Tibetan Pig PEDVs were originated from Sichuan region. This study will provide the theoretical basis for monitoring the new variation of PEDV.