RESUMO
We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.
Assuntos
Infecções por HIV/virologia , HIV-1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Kit de Reagentes para Diagnóstico , Carga Viral , HIV-1/genética , HumanosRESUMO
We developed a homogeneous format reverse transcription-polymerase chain reaction assay for quantitating hepatitis C virus (HCV) RNA based on the TaqMan principle, in which signal is generated by cleaving a target-specific probe during amplification. The test uses two probes, one specific for HCV and one specific for an internal control, containing fluorophores with different emission spectra. Titers are calculated in international units (IU)/ml by comparing the HCV signal generated by test samples to that generated by a set of external standards. Endpoint titration experiments demonstrated that samples containing 28 IU/ml give positive results 95% of the time. Based on these data, the limit of detection was set conservatively at 40 IU/ml. All HCV genotypes were amplified with equal efficiency and accurately quantitated: when equal quantities of RNA were tested, each genotype produced virtually identical fluorescent signals. The test exhibited a linear range extending from 64 to 4,180,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 21.6 to 30.4%, which implies that titers that differ by a factor of twofold (0.3 log10) are statistically significant (P = 0.005). The test did not react with other organisms likely to co-infect patients with hepatitis C and exhibited a specificity of 99% when evaluated on a set of samples from HCV seronegative blood donors. In interferon-treated patients, the patterns of viral load changes revealed by the TaqMan HCV quantitative test distinguished responders from nonresponders and responder-relapsers. These data indicate that the TaqMan quantitative HCV test provides an attractive alternative for measuring HCV viral load and should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.
Assuntos
Hepacivirus/genética , Hepatite C/virologia , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taq Polimerase/metabolismo , Genótipo , Hepacivirus/crescimento & desenvolvimento , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Humanos , Prognóstico , RNA Viral/genética , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga ViralAssuntos
Soronegatividade para HIV , Subpopulações de Linfócitos T , Síndrome da Imunodeficiência Adquirida/sangue , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Camarões , Países em Desenvolvimento , Feminino , Humanos , Contagem de Linfócitos , Masculino , Valores de Referência , Caracteres SexuaisRESUMO
This enzyme immunoassay (EIA) of human prolactin (hPRL) involves incubation of sample and anti-hPRL antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) in tubes coated with a second antibody to hPRL. The test can be performed within 60 min. No reaction of the antibodies with human placental lactogen and human somatotropin is detectable. The presence of detergent allows assay of both serum and plasma. Precision was improved by including polyethylene glycol in the reaction mixture. To optimize analytical recovery, we added protease inhibitor. Assay of the EIA standards shows good correlation with results for World Health Organization reference preparations. The measurable range is 1 to 400 micrograms/L. Intra- and interassay CVs are about 5%. Comparisons with two RIAs and two other EIAs show reasonably good correlations. The components of our EIA are stable for 18 months.
