| Sapovirus is a number in Caliciviridae family of positive sense single-stranded RNA viruses. Porcine sapovirus infection may cause diarrhea especially in piglets. Although porcine SaV was mainly detected in pigs, some porcine SaV strains were most closely related to human SaV, suggesting the possibility of a pig reservoir for human strains or vice versa. The outbreak of gastroenteritis in piglets in China suggested that procine SaV may bring potential harm to swine industry. It is important to develop a method for porcine sapovirus detection.Primers are devised according to SaV VP2 gene, and then VP2 gene was amplified by PCR, and inserted into the prokaryotic expression vector pET-30a. After identified by restrict enzyme and sequencing, the constructed recombinant expression plasmid was transformed into the receptive cells of E. coli BL21 and induced by IPTG with a finial concentration 1mmol·L-1, then the recombinant protein was expressed in the form of inclusion bodies. After the bacterium cell walls were disrupted by ultrasonication, the inclusion bodies of recombinant proteins were dissolved, and then were purified by Ni2+ affinity columns following with annealing. The expression protein can react with polyclonal antibody against SaV by Western-blot, sharing a good antigencity.An indirect enzyme-linked immnunostorbent assay (ELISA) based on the recombinant protein was developed. Using the purified recombinant proteins as coating antigen, the optimal coating condition was determined. It was shown that the optimal concentration of recombinant protein was 0.64μg·mL-1, coating time was 37℃for 1hour and 4℃overnight, 1% BSA was used as the best confining liquid, after being confined for 90 minutes at 37℃, testing sera dilution at l:50 and the sera reaction time is 1 hour at 37℃, the HRP-labeled rabbit anti-swine IgG dilution at l:2000 and the reaction time is 40 minutes at 37℃, the threshold for ELISA was 0.203. The recombinant protein showed no reaction with antiserum to HEV, EV9, NoV and S. suis 2. It indicated the indirect ELISA had good specificity for detection of SaV antibody in serum. 25 serum samples were detected by using this method, and the total masculine ratio was 88%. The result showed that the developed indirect ELISA had the advantages such as high sensibility, strong specificity and this method can be used to detect the porcine serum antibodies to SaV. It is hopefully to develop a SaV ELISA diagnostic kit based on the achievements derived in the present study, which provide scientific method for diagnosing SaV quickly in wide scope.The last, the primers and probes were designed and synthesized according to the conserved sequences of SaV, and a TaqMan real-time PCR assay were developed by optimizing the reaction conditions. Results showed that the real-time PCR assay could detect 16.1 copies·μL-1 of plasmid DNA, while the sensitivity of the routine RT-PCR was 1.61×103 copies·μL-1. 216 stool samples were then detected by the established quantitative PCR assay, and the results were compared with that of routine RT-PCR. It also showed that the sensitivity of established method was higher than that of the routine RT-PCR. Phylogenetic analysis indicated that all of the 4 SaV strains we had identified belonged to GIII, and shared 100% nucleotide homology with another Shanghai porcine SaV strain (FJ387164). The TaqMan real-time PCR assay, which is more specific, sensitive and accurate, can be used for the epidemiological investigation and diagnosis of porcine SaV infection. |
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