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Objective To study reaction principle of bilirubin vanadate oxidation method and bilirubin oxidase method through comparison of the determination results,and discuss similarities and difference between the two methods .Methods 310 ca-ses were measured and analyzed with each method.Abnormal samples were further investigated.Results Fortotal bilirubin, the regression equation obtained wasY=1.065 1X+1.197 2,the correlation coefficientr=0.997 0.For direct bilirubin of a-dults and children greater than 30 days,the regression equation wasY=0.945 9X+0.599 5 and the correlation coefficient r=0.994 4.For neonatal direct bilirubin,the regression equation wasY=0.410 4X+2.756 3 and the correlation coefficient r=0.883 5.The results from vanadate oxidation method were unacceptable for abnormal neonatal serum measurement after serial dilution.Conclusion The overall conclusions were that for the measurement of total bilirubin,and direct bilirubin for adults and children older than 30 days.The correlation between these two methods is in an acceptable range,for measure-ment of neonatal direct bilirubin,the correlation between thesetwo methods was not acceptable.It is not recommended to measure neonatal direct bilirubin by vanadate oxidation method.
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Objective To compare the degree of cell injury induced by 3D protein (SDLY11 and SDLY107) of enterovirus 71 (EV71) strains.Methods EV71 strains SDLY11 and SDLY107 were respectively isolated from children with mild and severe hand foot mouth disease.The target genes 11-3D-Flag and 107-3D-Flag were amplified by reverse transcription-polymerase chain reation (RT-PCR) and inserted into the eukaryotic expression vector pcDNA3.1.The recombinant plasmids 11-3D-Flag-pcDNA3.1 and 107-3D-Flag-pcDNA3.1 were transformed into Escherichia.coli DH5α, respectively, and were identified by enzyme digestion and sequencing.The recombinant plasmids were transfected into rhabdomyosarcoma (RD) cells, respectively.Expression of 3D protein was detected by indirect immunofluorescence assay and western blot.Cell injury induced by 3D protein was detected with lactate dehydrogenase (LDH) test, cell proliferation was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthiazolium bromide (MTT) test, and cell apoptosis was detected with Annexin-V and PI.Multiple comparisons among groups were analyzed using LSD-t test if multiple sets of variables were consistent with homogeneity of variance.If not, Dunnett T3 test was used.Results The 1 400 bp fragments were amplified by reverse tramscription (RT)-polymerase chain reaction (PCR), and the recombinant plasmids were digested by enzyme and the 1 400 bp and 5 400 bp fragments were obtained and identified.Gene sequencing showed that the sequences were consistent with the target genes.The specific fluorescence was observed by indirect immunofluorescence assay, and the western blot showed that the molecular weight of the target protein was 55×103.The LDH test showed that the A490 of SDLY11 3D protein transfection group (0.790±0.048) was higher than that of SDLY107 3D protein transfection group (0.641±0.018).The difference was statistically significant (t=5.14, P<0.05).The cell membrane damage caused by SDLY11 3D protein was more severe than SDLY107 3D protein.The MTT test showed that the A570 of SDLY11 3D protein transfection group (1.028±0.020) was lower than that of SDLY107 3D protein transfection group (1.081±0.002), and the difference was statistically significant (t=3.31, P<0.05).The effect on cell proliferation activity of SDLY11 3D protein was greater than SDLY107 3D protein.The results of Annexin-V/PI showed that the percentage of apoptotic cells of SDLY11 3D protein transfection group and SDLY107 3D protein transfection group were (1.471±0.246)% and (1.465±0.237)%, respectively, and the difference was not statistically significant (t=0.04, P=0.973).Conclusions Compared with the SDLY11 3D protein, SDLY107 3D protein induces slighter cell injury, has weaker effect on cell proliferation activity, and is more favorable for virus replication in cells.
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Objective To isolate enterovirus 71 from a death children,and analyze whether the neurovirulence was related to the variation of nucleotide and amino acid. Methods Enterovirus 71 was isolated from throat swabs which were colleted from Shandong Linyi People's Hospital. The full length genome was sequenced by amplification with RT-PCR and sequencing of 9 overlapped gene fragments covering full length of the genomes. The nucleotide and amino acid sequenced was aligned by BLAST, Bioedit and MEGA 4. Results A strain of enterovirus 71 was isolated and named as SDLY107. The full length was 7405 bp. The results of homology analysis of overall nucleotide sequence showed that strain Fuyang. Anhui. P. R. C/17.08/2 had highest homology (98.6%)with strain SDLY107, and the homology was 80.0% between strain SDLY107 with prototype strain BrCr/70,and 86. 5% between strain SDLY107 with nerve strain MS/87. Phylogenetic analysis showed that the phylogeny was close between SDLY107 with some isolated strains from Chinese Mainland, such as Beijing, Henan, Guangxi, Sbenzhen, Lanzhou, Fuyang, Chongqing and Zhejiang strains, which was clustered for C4 subtype. The results of amino acid sequence analysis showed that there were 2 mutations, E947D and K1873R, for strain SDLY107. Conclusion SDLY107 belonged to C4 subtype, amino acid mutations E947D and K1873R of which may be relevant to the pathogenicity of EV71.