Evaluation of two simplified methods for genotyping hepatitis C virus

Khan, Rahim; Tong, C. Y. William; Bloom, S.; Gilmore, Ian; Toh, Cheng Hock; Beeching, Nick

doi:10.1002/(sici)1096-9071(199705)52:1<35::aid-jmv7>3.0.co;2-s

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…False amplification was frequently observed, especially with the 1b, 1a, and 4 primer-probe sets. Similar "cross-reactive" patterns have been reported with other type-specific genotype amplification methods (11,13,15,17,19). In the majority of samples, the correct genotype amplification was sufficiently stronger than the cross-reactive amplification so the correct genotype could be determined.…”

Section: Discussionsupporting

confidence: 79%

“…Improved versions of the assays were described by Okamoto et al (25), Holland et al (15), Forns et al (11), and Ohno et al (22), which improved specificity and expanded the primer-probe sets to include the newly identified genotypes 4, 5, and 6. Several studies compared these type-specific assays to a variety of other genotyping methods (11,13,17,19). In each of these studies, results from the type-specific assays had good agreement with other methods but had somewhat high rates of mixed samples and samples that did not amplify.…”

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confidence: 99%

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Multiplex Real-Time Reverse Transcription-PCR Assay for Determination of Hepatitis C Virus Genotypes

et al. 2006

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A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n ‫؍‬ 110) and a subset that were incorrectly called 2a by the RFLP method (n ‫؍‬ 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.More than 60 genotypes and subtypes of hepatitis C virus (HCV) are distributed worldwide. Because therapeutic response rates differ by genotype, accurate and efficient genotype determination is critical to ensure correct treatment decisions.Initially, genotypes 1 and 2 were identified by the sequencing of multiple samples in the HCV core region (6,16,23). Analysis from samples worldwide and sequencing of additional genome areas including the 5Ј untranslated region (UTR), envelope, and NS5 regions identified genotypes 3, 4, 5, and 6. Sequence variation between genotypes, subtypes, and individual strains is greatest in NS5, less in the envelope and core, and least in the 5Ј UTR.Direct sequencing is the most accurate method for HCV genotyping. However, many other methods have been used because of the expense and technical difficulties of direct sequencing. The most frequently used methods in clinical laboratories are LIPA (line probe assay) and sequencing of the 5Ј UTR (both from Bayer Diagnostics). Both assays require two steps: generation of the PCR amplicon and then evaluation of the amplicon by either hybridization or sequencing. An additional disadvantage is the use of the 5Ј UTR, which is less informative for genotyping than are other, more variable regions of HCV.In contrast, type-specific PCR assays r...

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Section: Discussionsupporting

confidence: 79%

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confidence: 99%

Multiplex Real-Time Reverse Transcription-PCR Assay for Determination of Hepatitis C Virus Genotypes

et al. 2006

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“…Restriction fragment length polymorphism (RFLP) produces predicted patterns for each genotype. However, samples often do not match the published patterns, since restriction sites can be created or abolished with a single base mutation (Khan et al, 1997). A commercial kit (InnoLipa) has been introduced in Europe by Innogenetics (Zwijndre, Belgium) based on hybridisation of 5 NC biotin-labelled amplification products with 21 probes.…”

Section: Introductionmentioning

confidence: 99%

A real-time Taqman method for hepatitis C virus genotyping

Rolfe

Alexander

Wreghitt

et al. 2005

Journal of Clinical Virology

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“…The Amplicor HCV assay (Roche Diagnostic, Switzerland) was used for HCV RNA detection. HCV geneotyping was performed using a type-specific PCR primer method previously described (Khan et al, 1997). An in-house reverse transcription nested polymerase chain reaction (PCR) was used for the amplification of HGV RNA.…”

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confidence: 99%

Hepatitis G virus RNA and its relation to hepatitis C infection in adult haemophilic patients

et al. 1997

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Summary. The prevalence of hepatitis C virus (HCV) infection and hepatitis G virus (HGV) RNA were studied in 50 adult haemophilic patients who had received commercial clotting factors prior to 1980. HGV RNA was detectable in 6/ 50 patients (12%); 49/50 (98%) had antibody to HCV and 40/49 (82%) of these were viraemic with detectable HCV RNA; 5/6 patients with detectable HGV RNA had co-existing HCV infection and viraemia. The HGV PCR products from all six patients were directly sequenced and all were shown to be similar to that of HGV but more diverse from that of GB virus C. One patient who had persistent abnormal liver function tests had detectable HGV RNA but no evidence of hepatitis B or C. The presence of HGV RNA in the absence of hepatitis B and C infection indicates that this virus is capable of independent transmission. Independent response to interferon was demonstrated in one patient with co-infection who lost HGV but not HCV after interferon therapy.

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Evaluation of two simplified methods for genotyping hepatitis C virus

Cited by 5 publications

References 31 publications

Multiplex Real-Time Reverse Transcription-PCR Assay for Determination of Hepatitis C Virus Genotypes

Multiplex Real-Time Reverse Transcription-PCR Assay for Determination of Hepatitis C Virus Genotypes

A real-time Taqman method for hepatitis C virus genotyping

Hepatitis G virus RNA and its relation to hepatitis C infection in adult haemophilic patients

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