title: A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.
abstract: BACKGROUND
  Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment.
  
  
  OBJECTIVE
  Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control.
  
  
  METHODS
  A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination.
  
  
  RESULTS
  The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions.
  
  
  CONCLUSIONS
  In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube.
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey.
authors.forename: Ersin
authors.initials: E
authors.lastname: Karataylı
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey.
authors.forename: Yasemin Çelik
authors.initials: YÇ
authors.lastname: Altunoğlu
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey.
authors.forename: Senem Ceren
authors.initials: SC
authors.lastname: Karataylı
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey.
authors.forename: S Gökçe K
authors.initials: SG
authors.lastname: Alagöz
authors.affiliation: Ankara University, School of Medicine, Department of Gastroenterology, Ankara, Turkey.
authors.forename: Kubilay
authors.initials: K
authors.lastname: Cınar
authors.affiliation: Dicle University, School of Medicine, Department of Gastroenterology, Diyarbakır, Turkey.
authors.forename: Kendal
authors.initials: K
authors.lastname: Yalçın
authors.affiliation: Ankara University, School of Medicine, Department of Gastroenterology, Ankara, Turkey.
authors.forename: Ramazan
authors.initials: R
authors.lastname: Idilman
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey; Ankara University, School of Medicine, Department of Gastroenterology, Ankara, Turkey.
authors.forename: Cihan
authors.initials: C
authors.lastname: Yurdaydın
authors.affiliation: Ankara University, Institute of Hepatology, Ankara, Turkey. Electronic address: bozdayi@ankara.edu.tr.
authors.forename: A Mithat
authors.initials: AM
authors.lastname: Bozdayı
mesh_terms: D005260:Female; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods* / Q000592:standards
chemical_list: D012367:RNA, Viral
keywords: Armored RNA; HDV; Intrinsic control; Real time PCR