A novel nested reverse-transcriptase polymerase chain reaction method for rapid hepatitis C virus detection and genotyping.
OBJECTIVE
Rapid and specific detection of viral nucleic acid is increasingly important in the diagnosis of infectious diseases. The objective was to develop a rapid, efficient process of nucleic acid based detection of hepatitis C virus (HCV) infection for its diagnosis and treatment follow-up.


METHODS
A two-step nested reverse-transcriptase polymerase chain reaction (RT-PCR) has been standardised on a sample set of 125 individuals from different liver clinics in Kolkata. The method utilises a novel fast nested RT-PCR for HCV detection and genotyping from HCV infected patient plasma with high processivity.


RESULTS
The overall time required from ribonucleic acid (RNA) isolation to nested PCR amplified product detection is reduced to 42% when compared with conventional nested RT-PCR amplification. The method is sensitive as conventional PCR and detected all HCV RNA positive samples. Sequencing, phylogenetic analysis of the PCR amplified product by this method showed concordant genotypes with conventional PCR.


CONCLUSIONS
Though being a two-step process, this method is fast, cost-efficient, reliable and feasible for regular HCV RNA screening and apt even in resource limited settings. This method could be translated to regular nucleic acid screening for other infectious diseases as regular PCR regimen.
K
K
Saha
R
R
Firdaus
A
A
Biswas
A
A
Mukherjee
Department of Virology, I.C.M.R. Virus Unit, Kolkata, West Bengal, India.
P C
PC
Sadhukhan
D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction