RESUMEN
OBJECTIVE: To establish the national quantity standard of hepatitis B surface antigen according to the world health organization' s standard material and prepare the national liner reference panel for hepatitis B surface antigen. METHODS: Sera from hepatitis B patients and health blood donors in different areas were collected and detected by domastic HBsAg kits, anti-HBs kits, HBeAg kits, anti-HBe kits, anti-HBc kits and anti-HCV, and then confirmed by the kits produced by Abbott, which was approved by WHO. One serum with high concentration of HBsAg was calibrated with the standard sample of WHO. And then it was diluted by 1.5 fold as the liner HBsAg reference panel. RESULTS: The HBsAg concentration of one serum was 1226 IU/ml calibrated by 21 independent standardization measurements with 7 kinds of kits. The coefficient of variation of each calibration were less then 15%. A panel contained 8 serial dilutions was established as the national liner HBsAg reference panel. The permitted range of every dilution was stipulated and the stability of the panel was detected by accelerated test. CONCLUSIONS: The national quantity standard of hepatitis B surface antigen was established and the national quantitative reference panel for HBsAg which contains eight liner serum was developed.
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Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B/virología , Técnicas para Inmunoenzimas/métodos , Técnicas para Inmunoenzimas/normas , Juego de Reactivos para Diagnóstico/normas , China , Humanos , Estándares de ReferenciaRESUMEN
OBJECTIVE: To evaluate anti-HEV diagnostic kits by experimental infecting rhesus monkeys with HEV. METHODS: Eight rhesus monkeys were infected with genotype 1 and 4 HEV separately. The alanine aminotransferase (ALT) level of all monkeys were detected before and after the process of infection. HEV RNA in stool specimens was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Anti-HEV IgG in serum was detected by GL-IgG and WT-IgG. RESULTS: HEV RNA presented in the stool of all the 8 monkeys after infection. The ALT level of 1 monkey infected with genotype 1 HEV and 2 monkeys infected with genotype 4 HEV appeared abnormally after infection. Tested by GL-IgG, 2 of the 4 monkeys infected with genotype 1 HEV and 1 of 4 monkeys infected with genotype 4 HEV seroconverted to anti-HEV IgG. However, when tested by WT-IgG, all the infected monkeys seroconverted to anti-HEV IgG. The anti-HEV IgG tested by WT-IgG was positive during the whole observation period,and the anti-HEV IgG measured by GL-IgG only remained 12 weeks after infection. Detected by GL-IgG and WT-IgG, seropositive conversion of the anti-HEV IgG happened almost at the same time. CONCLUSION: Both GL-IgG and WT-IgG could detect the anti-HEV IgG of experimentally infected rhesus monkeys but the WT-IgG had a higher sensitivity for detection of anti-HEV IgG than
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Virus de la Hepatitis E/inmunología , Hepatitis E/inmunología , Hepatitis E/virología , Alanina Transaminasa/sangre , Animales , Modelos Animales de Enfermedad , Genotipo , Virus de la Hepatitis E/genética , Inmunoglobulina G/inmunología , Macaca mulatta , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To compare and analyze the sensitivity, specificity of 4 domestic ELISA kits for detection of hepatitis B virus (HBV) markers (HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc). METHODS: Five hundred and ninety four serum samples collected from patients with chronic hepatitis B and abnormal blood donors were detected for HBV markers and by 4 domestic ELISA kits. Samples with conflicting results by different diagnostic kits were retested. Samples with the HBsAg values close to the cut-off point were detected by Abbott HBsAg confirmation kit (Architect HBsAg confirm). Sensitivity of the kits was determined, using the national sensitivity reference panels for HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc. RESULTS: The rates of sensitivity on 4 domestic kits for detection of HBsAg were 4 to 10 times lower, and on the 4 domestic kits for detection of anti-HBs, HBeAg, anti-HBe and anti-HBc were 4 to 16 times lower, as compared to Abbott Architect kits. In addition, the domestic HBV ELISA kits had some false positive results. The total coincidence rates of HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc were 96.46%-98.15%, 94.28%-98.15%, 98.15%-99.49%, 90.07%-96.30%, 92.09%-96.80%, respectively. CONCLUSION: Both sensitivity and specificity of the domestically produced HBV ELISA kits should be improved.
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Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Hepatitis B/inmunología , Juego de Reactivos para Diagnóstico/clasificación , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Humanos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To evaluate the multiplex nucleic acid testing (NAT) assays for HBV, HCV and HIV in detecting HBV DNA in plasma samples. METHODS: 534 plasma samples collected form several areas were detected with Abbott Architect i2000 HBsAg, ani-HBs, HBeAg, anti-HBe, anti-HBc and anti-HBc IgM diagnostic kits. HBV DNA levels of those samples were detected with Roche COBAS AmpliPrep/COBAS TaqMan HBV Test. Two kinds of multiplex NAT assays for HBV, HCV and HIV were used to test HBV DNA of those 534 samples. Results of serology-markers and quantitative HBV DNA levels with results of NAT were compared. RESULTS: HBV DNA was positive in all 81 HBsAg, HBeAg and anti-HBc positive samples, detected by both of NAT assays. HBV DNA was positive in 11 and 19 of 200 HBsAg negative samples when detected with the two kinds of NAT assays separately. Compared with the quantitative results detected by Roche COBAS AmpliPrep/COBAS TaqMan HBV Test, the HBV DNA positive rates were 96.9% and 94.3% in 193 samples of HBV DNA levels over 500 IU/ml while 40.2% and 45.3% in 117 samples of HBV DNA levels below 500 IU/ml while 99.3% and 96.0% in 151 samples of DNA negative HBV. CONCLUSION: There are some occult low level HBV DNA carriers with HBsAg negative results in China. NAT assays for HBV, HCV and HIV may be useful to improve the transfusion safety.
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Donantes de Sangre , ADN Viral/sangre , Virus de la Hepatitis B/genética , ADN Viral/genética , Humanos , Pruebas Serológicas/métodosAsunto(s)
Proteínas Recombinantes de Fusión , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/inmunología , Clonación Molecular , Escherichia coli/genética , Hepacivirus/genética , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas no Estructurales Virales/genéticaRESUMEN
Synthesis Large Fragment DNA using PCR (SLFD PCR) is a useful method to synthesize large fragment DNA. Use a known 500 approximately 600 basepair DNA fragment as PCR template, a series of 5' terminal primers are designed, and these primers are overlap one by one from 5' terminal to 3' terminal, the net DNA is just what you want to synthesize. The last 3' terminal primer of these primers has a BamH I site, and behind the BamH I site there are 15 bp overlap the 5' terminal of the template. Another downstream primer complement the 3' terminal of the template, and has a BamH I site too. PCR begin using the innerest 5' terminal primer and the downstream primer. After 10 cycles of PCR, use the product of the PCR as the template of next round PCR, but this time the upstream primer change to the 5' terminal outer primer . So do the PCRs, till all the 5' terminal primers take part in the PCR. Clone the final PCR product and BamH I cut the original DNA template, The DNA synthesis complete. It's a useful method to synthesize 100 approximately 200 bp DNA fragment, even more long DNA fragment.