Relevance of a Full-Length Genomic RNA Standard and a Thermal-Shock Step for Optimal Hepatitis Delta Virus Quantification

TY - JOUR

T1 - Relevance of a Full-Length Genomic RNA Standard and a Thermal-Shock Step for Optimal Hepatitis Delta Virus Quantification

AU - Homs, Maria

AU - Giersch, Katja

AU - Blasi, Maria

AU - Lütgehetmann, Marc

AU - Buti, Maria

AU - Esteban, Rafael

AU - Dandri, Maura

AU - Rodriguez-Frias, Francisco

N1 - Copyright © 2014, American Society for Microbiology. All Rights Reserved.

PY - 2014/9/1

Y1 - 2014/9/1

N2 - Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to -80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification.

AB - Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to -80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification.

U2 - 10.1128/JCM.00940-14

DO - 10.1128/JCM.00940-14

M3 - SCORING: Journal article

C2 - 24989607

VL - 52

SP - 3334

EP - 3338

JO - J CLIN MICROBIOL

JF - J CLIN MICROBIOL

SN - 0095-1137

IS - 9

ER -