sybr-green-i and Hepatitis-C

Detection of the HCV genome is crucial for diagnosis of HCV infection and for monitoring the efficacy of interferon treatment for patients with HCV. We developed a convenient screening test for HCV genotypes 1 and 2 based on the melting curve analysis with SYBER green I. Serum samples were drawn from 114 patients with known chronic HCV infection confirmed to be antibody-positive by immunoblot assay. A characteristic melting profile for each genotype was obtained by monitoring the fluorescence as the temperature increases through the melting point of the PCR product. Serum samples with HCV-RNA genotype (1b, 2a and 2b) were analyzed every test as standard samples and the genotype of unknown samples was determined by the comparison with the melting point of standard samples. Serum samples with known HCV-RNA genotype (1b, 2a and 2b) and HCV-RNA-negative sample were tested using the Light cycler system. The melting curve analysis indicated that melting points are 93.08 +/- 0.56 degrees C for genotype 1b (n = 63), 91.08 +/- 0.49 degrees C for genotype 2a (n = 33), and 91.77 +/- 0.28 degrees C for genotype 2b (n = 18). The melting points for genotypes 1b, 2a, and 2b differed by approximately 1 degree C in each other. The genotype was determined for all samples using Okamoto's method and Light cycler system, and both systems produced absolutely identical results for all the samples studied. Sixty-three of 114 were genotype 1b, 33 samples were genotype 2a, and 18 were genotype 2b. This melting curve analysis is a rapid and convenient screening test for differentiation of HCV genotypes 1 and 2.

Topics: Benzothiazoles; Diamines; Genotype; Hepacivirus; Hepatitis C; Humans; Organic Chemicals; Polymerase Chain Reaction; Quinolines; RNA, Viral

We intend to design and validate a low-cost assay for the detection of hepatitis C virus (HCV) RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours.. A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DNA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay.. The minimum detection level of our assay was less than 50 IU/mL. The results on 100 plasma samples were comparable with commercial assays.. This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications.

Topics: Benzothiazoles; Diamines; Fluorescent Dyes; Hepacivirus; Hepatitis C; Humans; Limit of Detection; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Time Factors

Dried blood spot (DBS) is a reliable method of blood collection used for the diagnosis of several human diseases. DBS is particularly useful for diagnosing children and for the screening of high-risk populations especially in countries where health facilities are not readily accessible. This report describes a qualitative SYBR Green-based real-time multiplex RT-PCR for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genomes in DBS. Specific viral amplicons were identified in the same sample by their distinctive melting temperatures. The analysis of scalar concentrations of the reference samples indicated that this multiplex procedure detects at least 2500 copies/ml of HCV and 400 copies/ml of HIV-1. HIV-1 and HCV viral loads in 20 patients infected with HIV-1 and/or HCV and in 5 healthy blood donors were also tested, confirming the sensitivity and specificity of the assay. This method may represent a reliable alternative for the detection of HIV-1/HCV co-infection, in rapid and relatively inexpensive screening programmes.

Topics: Adult; Benzothiazoles; Blood; Desiccation; Diamines; Hepacivirus; Hepatitis C; HIV Infections; HIV-1; Humans; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Specimen Handling; Staining and Labeling; Time Factors; Transition Temperature; Young Adult

Qualitative detection of negative hepatitis C virus (HCV) RNA has been used widely to demonstrate HCV replication. However, relative quantitation of both positive and negative HCV RNA strands has never been reported for studying viral genome replication. A strand specific real-time PCR carried out in the highly conserved 5'-non-coding region of HCV genome and monitored either by the DNA binding dye SYBR Green I or by molecular beacons is described. Using these techniques, it was found that negative HCV RNA strand was a 100-1000 times less abundant than the positive strand in the liver of HCV infected patients.

Topics: 5' Untranslated Regions; Benzothiazoles; Diamines; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Liver; Molecular Probes; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reproducibility of Results; RNA, Viral; Viral Load; Virus Replication

Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. We needed to develop a convenient screening test for HCV genotypes 1 and 2.. We tested 55 patients with known chronic HCV infection. Viral RNA was extracted from serum samples using an automatic viral RNA purification system, and HCV genotypes were determined by reverse transcriptase-polymerase chain reaction using LightCycler melting curve analysis with SYBR Green I.. HCV RNA was detected in all samples and each genotype was determined. The mean (standard deviation) melting temperatures for subtypes 1b (n = 32), 2a (n = 15) and 2b (n = 8) were 93.14 degrees C (0.51 degrees C), 91.08 degrees C (0.49 degrees C) and 91.77 degrees C (0.27 degrees C), respectively. Genotypes 1 and 2 were differentiated within 3 h by this method.. Our melting curve analysis is a rapid and convenient screening test for differential identification of HCV genotypes 1 and 2.

Topics: Benzothiazoles; Diamines; DNA Primers; DNA, Complementary; Genome, Viral; Genotype; Hepacivirus; Hepatitis C; Humans; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sequence Analysis, DNA; Virulence