ABSTRACT
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.
ABSTRACT
To understand the genetic diversity of porcine deltacoronavirus(PDCo V) in Guangxi Province, clinical diarrhea samples were collected from suspected piglets in Guangxi Province from2017 to 2019, detected by RT-PCR for PDCoV, and the positive samples were used for amplification and sequence of S, M, N genes. Finally, 16 S, M and N gene sequences of PDCoV were obtained. Homology analysis showed that the S, M, N gene nucleotide identity among Guangxi strains were 95.8% -99.9%, 95.9%-100% and 97.9%-99.9%, respectively. The nucleotide identity of S, M and N genes among Guangxi strains and other reference strains were 95.1%-100%, 95.0%-100%and 96.3%-99.9%, respectively. Sequence alignment showed that S1 protein existed amino acid mutations and insertions, and there were some variations among different epidemic strains. Phylogenetic trees based on S, M and N genes obtained similar topological diagram and all strains could be divided into Group I, Group II and GroupIII, of which Group I came from USA, Japan and Korea, Group II came from China, and Group III came from China, Vietnam, Laos and Thailand. Most strains from Guangxi Province distributed in Group II, individual strain distributed in Group III and some strains formed a single small branch. The evolutionary rates of S, M and N genes of Guangxi strains and other reference strains were 2.57 x 10-4, 2.07 x 10-4, 1.70 x 10-4 substitutions/site/year, respectively, showing that the evolutionary rate of S gene was the fastest. The results indicated that the S, M, N genes of PDCo V strains from Guangxi Province had some variations and existed genetic diversity.
ABSTRACT
To establish a rapid method for simultaneous detection of senecavirus A (SVA) and O, Asia I, A serotypes of foot-and-mouth disease virus (FMDV), four pairs of specific primers were designed to amplify SVA 3D gene (157 bp), FMDV VP1 gene of serotype O (240 bp), Asia I (460 bp) and A (320 bp), respectively. After optimizing the primer concentration, annealing temperature and other reaction conditions, we established a sensitive, specific and reproducible method for simultaneous detection of SVA and O, Asia I, A serotypes of FMDV. The method specifically detects SVA and O, Asia I, A serotypes of FMDV without reacting with CSFV, PRRSV, PEDV, PRV, PPV, PCV2 and PCV3. The lowest detection limits of SVA and O, Asia I, A serotypes of FMDV were all 2.5x102 copies/L. Furthermore, consistent results were produced under uniform reaction conditions. We used the method to detect 30 field samples collected from Guangxi province in 2019, results showed the positive rates of SVA and O serotype of FMDV were 16.67% and 63.33% respectively, while Asia I, A serotypes of FMDV was not detected. Meanwhile, by testing the same field samples using the state standard diagnostic method for FMDV and a reported nested RT-PCR method for SVA, we found that the coincidence rate of established method and the comparative methods was 100%. The results indicated that the established multiplex RT-PCR method, which had high sensitivity, specificity and reproducibility, could be used for differential detection of SVA and O, Asia I, A serotypes of FMDV.