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ISSN 1561-3054 (Electronic)

2012, Number 3

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Rev Cubana Med Trop 2012; 64 (3)

Real-time polymerase chain reaction assay for Hepatitis B virus DNA quantification

Rodríguez LLA, Montalvo VMC, Sariego FS, Bello CM, Mora LE, Kourí CV, Martínez RPA, Sánchez WM, Marrero B

Language: Spanish
References: 25
Page: 290-303
PDF size: 190.74 Kb.


ABSTRACT

Introduction: viral DNA levels in serum samples are a useful marker to monitor the disease progression and the treatment response in patients with chronic hepatitis B. Commercial kits for this purpose are available, but they are considerably expensive.
Objectives: to evaluate the analytical performance of a real-time polymerase chain reaction (RT-PCR) assay for Hepatitis B virus DNA quantification.
Methods: specific primers to the gene C and TaqMan chemistry in a LightCycler 1.5 equipment was used. A standard curve was made and evaluated. Two hundred and seventy-two serum samples were used to assess the clinical and analytical specificity, the genotypic accuracy and specificity, the intra-assay and interassay coefficients of variation and the comparison with a commercial assay and with the qualitative PCR.
Results: the standard curve showed a strong linear correlation (r= -1) and low error values in the tested target DNA concentration. Analytical and clinical specificities were 100 %. Genotype accuracy and specificity showed that the differences between the results obtained by RT-PCR assay and those of the reference assay were less than 0.5 Log10. The 95% HBV DNA detection end-point assessed by Probit analysis was 16.41 IU/µL with a dynamic range of quantification of 108 IU/mL. Intra-assay and interassay coefficients of variation ranged from 0.16 to 1.45 % and 0.9 to 2.62 % respectively. The RT-PCR assay correlated well with those from a commercial assay (r= 0.964 and r2= 0.929) and with the HBV qualitative PCR, thus confirming its better sensitivity and advantages.
Conclusions: the RT-PCR assay is well suited to monitoring HBV DNA levels showing to be sensitive, specific and reproducible. Its application in the clinical practice ensures a better diagnosis and management of patients with chronic hepatitis B in Cuba.


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