Development Of Indirect ELISA And Real-Time PCR Assay For Detection Of Hpev

Primers were devised according to HPeV VP1 gene for amplifying 696bp aimed DNA fragments with RT-PCR, which were then cloned into the prokaryotic expression vector pET-28a. After identified by restriction endonuclease digestion analysis and sequencing, the recombinant plasmid was transformed into the E.coli Rosetta (DE3) strain and induced by IPTG, SDS-PAGE indicated that the target protein was insoluble in the form of inclusion bodies. The inclusion bodies were dissolved in 6M GuHCl and purified by Ni2+ affinity columns, renaturation proceeded under the condition of 4℃.The expression protein can react with HPeV-positive serum by Western blot assay, showing a good antigenicity.Using the purified recombinant protein as the coating antigen, an alternative indirect enzyme-linked immunosorbent assay (ELISA) for detection of HPeV antibody was developed. It was shown that the optimal antigen coating concentration was 3.7μg?ml-1, the optimal condition for coating was 37℃for 2h and 4℃overnight, 5% skimmed milk was chosen as the best confining liquid, the optimal serum dilution was selected 1:400 and the reaction time was 1h at 37℃, the HRP-labeled goat anti-human IgG dilution was at 1:5000, and the final substrate chromogenic time was 10min.The threshold for ELISA was calculated to be 0.1701.The results of repeatability and specificity test showed a good reproducibility, and there was no cross reaction existing in ELISA test. 257 clinical serum samples were detected by this assay , seroprevalence rate for HPeV was found to be 73% (187/257) in children(～6-year old) in Shanghai of China. According to the genome sequences of the eight HPeV genotypes available in GenBank,a pair of specific primers targeting the conserved 5'untranslated region (5'UTR) were designed. By optimizing the reaction parameters, a SYBR Green I real-time PCR assay was developed. Results showed a good linear relationship between initial templates numbers and Ct values, with an excellent correlation coefficient (r2=0.999) and specific melting curve. The assay had a detection threshold of 60 copies/μl of initial templates, which was 100 times more sensitive than conventional PCR. Moreover, the variability of repetitive Ct value less than 1% exhibited a good reproducibility. 156 fecal samples were detected by this established assay, and the result showed a higher sensitivity compared with routine nested RT-PCR. The high sensitivity, specificity and reproducibility of this assay, together with the relatively simple procedure, indicated its potential use in HPeV clinical diagnosis and epidemical investigation.