DEVELOPMENT OF A MULTIPLEX REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION ASSAY FOR RAPID DISCRIMINATION BETWEEN HEPATITIS E VIRUS GENOTYPES 3 AND 4

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  • E型肝炎ウイルスの遺伝子型3型株および4型株迅速鑑別検査法の開発

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Abstract

<p>Hepatitis E virus (HEV) is the causative agent of hepatitis E. Highly pathogenic HEV genotype 4 strains are frequently detected, with two cases of transfusion-transmitted hepatitis E confirmed in 2002 and 2004 in Hokkaido, Japan. Therefore, HEV nucleic acid amplification tests (NATs) have been implemented on a trial basis in Hokkaido since January 2005. HEV RNA-positive samples are quantitatively assayed for HEV RNA and are genotyped using phylogenetic analysis, which takes several days. We developed a multiplex real-time reverse transcription (RT) polymerase chain reaction (PCR) assay to rapidly discriminate between genotypes 3 and 4 (D-PCR). Different fluorescent dyes were used to label specific probes to genotypes 3 and 4. This method required only approximately 4.5 hours for genotyping by D-PCR, and quantification of HEV RNA could be performed simultaneously. The average sensitivity of D-PCR was inferred to be 38 IU/ml for genotype 3 and 68 IU/ml for genotype 4 when using 1 ml of plasma. The genotypes determined by D-PCR for 297 of 340 HEV RNA-positive samples were consistent with those determined by phylogenetic analysis. The D-PCR method established in the present study promptly provides blood donors and medical institutions with useful medical information related to hepatitis E.</p>

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