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Publication type: Journal Article
Document type: Full Paper

Year: 2011

Author(s): Troxler, S; Marek, A; Prokofieva, I; Bilic, I; Hess, M

Title: TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

Source: J Clin Microbiol. 2011; 49(4):1339-1346



Authors Vetmeduni Vienna:

Bilic Ivana
Hess Michael
Marek Ana
Prokofieva Irina
Troxler Salome

Vetmed Research Units
University Clinic for Poultry and Fish Medicine, Clinical Unit of Poultry Medicine


Abstract:
Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

Keywords Pubmed: Animals
Chickens
Conserved Sequence
DNA Primers/genetics
Hepatitis, Viral, Animal/diagnosis*
Hepatitis, Viral, Animal/virology*
Hepevirus/genetics
Hepevirus/isolation & purification*
Molecular Sequence Data
Oligonucleotide Probes/genetics
Poultry Diseases/diagnosis
Poultry Diseases/virology
RNA Virus Infections/diagnosis
RNA Virus Infections/veterinary*
RNA Virus Infections/virology
RNA, Viral
Reverse Transcriptase Polymerase Chain Reaction/methods*
Reverse Transcriptase Polymerase Chain Reaction/standards
Sensitivity and Specificity
Sequence Analysis, DNA
Viral Proteins/genetics
Virology/methods*
Virology/standards


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