ABSTRACT
Purpose: 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. Materials and 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. Conclusion: 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.
ABSTRACT
Objective To explore the relationship between viral encephalitis(VE)and Borna disease virus(BDV)infection,and discuss the clinical features of viral encephalitis patients infected by BDV.Methods The p24 gene fragment of BDV in cerebrospinal fluid mononuclear cells(CSFMC)from 32 patients with viral encephaliris and 34 healthy individuals were examined by fluorescence quantitative nested reverse transcriptase polymerase chain reaction(FQ-nRT-PCR),in which, β-actin was used as internal reference. The clinical manifestations of patients with positive BDV p24 were watched. And the gene sequence,homology and amino acid sequence of positive products were analyzed. Results The positive rate(4/32,12.5%)of BDV p24 in CSFMC of patients with viral encephalitis was significantly higher than that of healthy individuals(0/34,0%),P＜0.05,and the numbers of copies of BDV p24 in CSFMC of VE patients were more than 102/μl. Compared with the sequences of the standard strain Ⅴ and the virus strain H1766 of BDV from horse supplied by GenBank,the homology of the gene sequence of positive product of CSFMC was 95.35% and 98.84% with gene mutation in 4 sites(nt1649 T→C, nt1656 G→A,nt1670 C→T, nt1676 C→T). The genetic relationship between the positive product and the virus strain BDV from horse was the nearest. The clinical features of VE patients with positive BDV p24 were mainly by mental and behavior disorders. Conclusion The occurrence of VE patients in the city of Zunyi in Guizhou province may be associated with BDV infection. The clinical features of VE patients with positive BDV p24 were mainly mental and behavior disorders.