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Human cosavirus (HCoSV) is a small RNA virus family (Picornaviridae), is a single-stranded RNA virus. To date, a total of five seed virus isolated, respectively, for A, B, C, D, E (Human cosavirus AE). Although the relationship between HCoSV clinical symptoms are not clear, but the detection rate is relatively high, it may occur with other enteric viruses mixed infection or disease-induced factors, pose a potential threat to human health. Therefore, the establishment of a sensitive, specific PCR method HCoSV significance. Amplified from the establishment of the of HCoSV nested PCR detection method and initial application, the viral genome sequence and analysis of the VP3 protein expression and purification system, the results are as follows: The study is based on the GenBank published HCoSV sequence 5 'end of the non- coding region (5'UTR) specific primers were designed to establish the HCoSV nested PCR detection method, established through a series of optimization of reaction conditions, specificity experiments and sensitivity testing, a fast, simple, and accurate nested PCR method for detecting HCoSV. PCR reaction the objective fragment encoding the 5 'end non-coding region (5'UTR) of the RNA fragments, the size of 414bp. System optimization 50ul reaction, the annealing temperature was 55 ℃, the final concentration of primers for 1umol / L, MgCl2 concentration was 1.25mmol / L, the Taq enzyme concentration of 0.1 U / ul. Swine hepatitis E virus (Hepatitis E virus HEV) Porcine enterovirus type 9 (Enterovirus EV9), of astrovirus (Human Astrovirus HAstV), Boca virus (Human bocavirus HBoV), Seve virus (Saffold virus, SAFV) no specific reaction. Nested PCR method to establish initial detection fresh fecal samples collected in the Shanghai area 248 (188 children with diarrhea and 60 were healthy children). The results showed: diarrhea in children, a total of 6 were positive, the positive rate of 3.2%, five healthy children were positive, the positive rate was 1.6%, the phylogenetic tree and homology analysis found a total of seven positive samples for HCoSVA the species (HCoSV-A), indicating that the strains HCoSV-A may be popular for the Shanghai region. The experiments show that the to establish nested PCR method can be used for the clinical detection of fecal samples, specific, sensitive, accurate, concise, the establishment of the method to provide a scientific basis for the rapid diagnosis of clinical infection by HCoSV. Known sequence information HCoSV GenBank were designed 10 pairs of primers to amplify the full genome sequence of a virus with RT-PCR and Genome Walking, and analysis of the gene sequences of the strains of the virus. The results showed that: a virus genome length of approximately 7262nt, comprising a multimeric protein 2125aa. VP3 gene fragment was amplified, cloned into the prokaryotic expression vector pET-30a, after digestion, DNA sequencing, the recombinant plasmid was transformed into E. coli BL21, with a final concentration of 1 mmol? L-1 IPTG was induced expression products exist in the form of inclusion bodies. Ultrasonic lysis bacterial inclusion bodies dissolved, then Ni-NTA column purified recombinant proteins, and the purified protein refolding, the acquisition of the whole genome sequence of the VP3 protein expression to the next step protein functional genomics research and ELISA for the detection of the establishment of the foundation.
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