Melting Curve Analysis for the Screening of Hepatitis B Virus Genotypes A, D and F in Patients From a General Hospital in Southern Brazil

Abstract

-Context -Hepatitis B virus (HBV) can cause fulminant hepatitis, cirrhosis and hepatocellular carcinoma, and is one of the most common causes of acute and chronic liver failure. The genetic variants of HBV can be decisive for the evolution of these diseases as well as for the election of therapy. Objectives -The aim of this study was to evaluate and standardize an in house methodology based on the analysis of the melting curve polymerase chain reaction (PCR) of real-time (qPCR) to screen for genotypes A, D and F of HBV in patients from a hospital in Rio Grande do Sul, Brazil. Methods -We evaluated 104 patients presumably with HBV chronic infection. Viral DNA was extracted from plasma and viral genotypes and different mutations were determined using PCR-based protocols. Results -A PCR-based methodology was standardized for the analysis of genotypes A, D and F of HBV. The technique was based in a nested PCR with the final step consisting of a multiplex real-time PCR, using the melting curve as a tool for the differentiation of fragments. A higher frequency of genotype D (44.4%), followed by genotype A (22.2%) and genotype F (3.7%) was observed. Conclusion -The standardized assay, a nested PCR-multiplex qPCR using specific primers, provides a rapid and accurate method for the differentiation of HBV genotypes that are more frequent in Southern Brazil -A, D and F. This method can be applied in the clinical practice.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

“… Moraes et al (1996) found genotypes A, D, and F in the state of Rio de Janeiro. These same genotypes were also described in the populations of the city of Goiânia, state of Goiás, and of the state of Santa Catarina ( Teles et al 1999 , Carrilho et al 2004 , Viana et al 2005 , Alcalde et al 2009 , Becker et al 2013) .…”

“… Becker et al (2013) standardised an in-house rtPCR for genotyping different strains of HBV. These authors conducted a study employing set of primers specifically for genotype A, D, and F because of these are more common in our country.…”