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Comparison of hepatitis C virus genotyping by 5' noncoding region- and core-based reverse transcriptase PCR assay with sequencing and use of the assay for determining subtype distribution in India.

ABSTRACT: Phylogenetic analysis of the sequences of the 5' noncoding regions (5'NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify them further into subtypes as 1b (number of isolates [n] = 10), 1a (n = 6), 3a (n = 9), 3g (n = 14), 3f (n = 1), and 4d (n = 3). Three new subtypes were identified for the first time and designated as 3i (n = 5), 3j (n = 2), and 6l (n = 1). Sequencing the 5'NCR could differentiate HCV types, whereas classification at the level of subtype was possible with sequence analysis of the core region.

SUBMITTER: Lole KS

PROVIDER: S-EPMC262521 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Comparison of hepatitis C virus genotyping by 5' noncoding region- and core-based reverse transcriptase PCR assay with sequencing and use of the assay for determining subtype distribution in India.

Lole Kavita S KS Jha Jyotsna A JA Shrotri Sandhya P SP Tandon Badri N BN Prasad V G Mohan VG Arankalle Vidya A VA

Journal of clinical microbiology 20031101 11

Phylogenetic analysis of the sequences of the 5' noncoding regions (5'NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify th ...[more]

PMID: 14605173

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Project description:Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

Dataset Information

Comparison of hepatitis C virus genotyping by 5' noncoding region- and core-based reverse transcriptase PCR assay with sequencing and use of the assay for determining subtype distribution in India.

Publications

Comparison of hepatitis C virus genotyping by 5' noncoding region- and core-based reverse transcriptase PCR assay with sequencing and use of the assay for determining subtype distribution in India.

Similar Datasets

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