Dataset Information

Determination of human rotavirus VP6 genogroups I and II by reverse transcription-PCR.

ABSTRACT: Based on nucleotide sequence and phylogenetic analysis of the partial VP6 genes, group A rotaviruses can be mainly differentiated into two genogroups. In this study, a method employing reverse transcription-PCR (RT-PCR) and degenerate primers was established to assign the VP6 genogroup. VP6 genogroup I and genogroup II could be determined according to the sizes of the amplicons: 380 and 780 bp, respectively. The VP6 genogroup of human reference strains of G1 to G4 and G9 types and RotaTeq vaccine strains could be properly assigned by RT-PCR. Eighty rotavirus-positive fecal samples were subjected to enzyme-linked immunosorbent assay (ELISA), RT-PCR, and sequencing of the partial VP6 gene for subgroup and genogroup determination. The results correlated well among these three methods, except for seven samples whose subgroups could not be determined by ELISA. VP6 genogroups of another 150 rotavirus strains recovered between 1981 and 2005 were determined by RT-PCR and sequencing, and the same results were obtained by these two methods. Furthermore, an additional 524 rotavirus-positive fecal samples were tested by RT-PCR, and the VP6 genogroups could be easily determined. The RT-PCR assay developed here provided a reliable and convenient method for assigning the VP6 genogroups of human rotaviruses with a wide range of genetic variation.

SUBMITTER: Lin YP

PROVIDER: S-EPMC2566130 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Determination of human rotavirus VP6 genogroups I and II by reverse transcription-PCR.

Lin Yi-Pei YP Kao Chuan-Liang CL Chang Sui-Yuan SY Taniguchi Koki K Hung Pei-Yung PY Lin Hsueh-Ching HC Huang Li-Min LM Huang Hsueh-Hung HH Yang Jyh-Yuan JY Lee Chun-Nan CN

Journal of clinical microbiology 20080730 10

Based on nucleotide sequence and phylogenetic analysis of the partial VP6 genes, group A rotaviruses can be mainly differentiated into two genogroups. In this study, a method employing reverse transcription-PCR (RT-PCR) and degenerate primers was established to assign the VP6 genogroup. VP6 genogroup I and genogroup II could be determined according to the sizes of the amplicons: 380 and 780 bp, respectively. The VP6 genogroup of human reference strains of G1 to G4 and G9 types and RotaTeq vaccin ...[more]

PMID: 18667595

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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

Determination of human rotavirus VP6 genogroups I and II by reverse transcription-PCR.

Publications

Determination of human rotavirus VP6 genogroups I and II by reverse transcription-PCR.

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