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Titolo:
Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay
Autore:
Jardi, R; Rodriguez, F; Buti, M; Costa, X; Cotrina, M; Valdes, A; Galimany, R; Esteban, R;
Indirizzi:
Hosp Univ Valle Hebron, Dept Biochem, Barcelona, Spain Hosp Univ Valle Hebron Barcelona Spain , Dept Biochem, Barcelona, Spain Hosp Univ Valle Hebron, Dept Hepatol, Barcelona, Spain Hosp Univ Valle Hebron Barcelona Spain , Dept Hepatol, Barcelona, Spain
Titolo Testata:
JOURNAL OF VIRAL HEPATITIS
fascicolo: 6, volume: 8, anno: 2001,
pagine: 465 - 471
SICI:
1352-0504(200111)8:6<465:QDOHBV>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
HBV-DNA; SIGNAL AMPLIFICATION; DETECTION SYSTEM; INFECTION; QUANTIFICATION; BLOOD;
Keywords:
chronic hepatitis; HBV DNA; hepatitis B virus; lamivudine; LightCycler; real-time PCR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Clinical Medicine
Citazioni:
22
Recensione:
Indirizzi per estratti:
Indirizzo: Jardi, R Hosp Gen Valle Hebron, Serv Bioquim, Paseo Valle Hebron S-N, Barcelona 08035, Spain Hosp Gen Valle Hebron Paseo Valle Hebron S-N Barcelona Spain 08035
Citazione:
R. Jardi et al., "Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay", J VIRAL HEP, 8(6), 2001, pp. 465-471

Abstract

A sensitive and accurate HBV DNA quantification assay is essential for monitoring hepatitis B virus (HBV) replication. This study evaluated a real-time PCR method performed in the LightCycler(TM) analyser for quantitative HBV DNA assay. HBV DNA results with this method were compared with those obtained using a branched-chain DNA (bDNA) solution hybridization assay. Real-time PCR was performed using two adjacent fluorescently labelled probes and primers corresponding to the HBV core gene. The same standard employed in the bDNA assay was used for calibration. Serum samples came from 193 HBV surface antigen (HBsAg)-positive patients (34 HBV e antigen (HBeAg)-positive and 93 with antibody to HBeAg (anti-HBe)), and 66 asymptomatic HBV carriers. In addition, we analysed serum samples from 8 anti-HBe-positive patients who had been receiving lamivudine treatment for more than three years. A linear standard curve was seen in the range from 10(3) to 10(8) copies/mL. In the reproducibility analysis, intra-assay coefficient of variation (CVs) attwo known HBV DNA concentrations were 4% and 2% and interassay CVs were 6%and 4%. The median of serum HBV DNA by realtime PCR was 9.2 x 10(8) copies/mL in MAg-positive patients with persistently elevated alanine aminotransferase (ALT) levels, 1.3 x 10(7) copies/mL in anti-HBe-positive cases with persistently elevated ALT levels, 3.7 x 10(4) copies/mL in anti-HBe-positivepatients with fluctuating ALT levels and 10(4) copies/mL in asymptomatic HBV carriers. The differences in HBV DNA levels among the various groups studied were statistically significant (P<0.05). The cut-off between chronic hepatitis patients and asymptomatic carriers was found to be at a serum HBV DNA concentration of 5 x 10(4) copies/mL. Of the 109 serum samples with a viral load < 7.5 x 10(5) (negative by bDNA assay) 44 (40%) were positive by real-time PCR: 24 (56%) chronic hepatitis and 20 (33%) asymptomatic carriers. There was a positive association between HBV DNA levels determined by real-time PCR and ALT levels (P<0.05), which was not observed with the bDNA assay for HBV DNA quantification. At 12 months of lamivudine treatment, 6 patients (75%) showed HBV DNA levels < 5 x 10(4) copies/mL (range < 10(3) -2 x 10(3)), significantly lower than at baseline. At 36 months, 2 of 8 (25%) showed HBV DNA levels persistently lower than 5 x 10(4) copies/mL (1.7 x 10(3), 6 x 10(3)). The LightCycler quantitative real-time PCR is a practical,sensitive, reproducible single-tube assay with a wide dynamic range of detection. The assay is automatic except for DNA extraction and the running time is only 70 min. The LightCycler real-time PCR is useful for identifying different states of HBV infection and for evaluating the efficacy of viral therapy.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/01/20 alle ore 01:52:39