Quantitative polymerase chain reaction for hepatitis B virus DNA.
To monitor and compare the effect of drugs on hepatitis B virus (HBV) replication in hepatitis patients, an accurate quantitative method with high sensitivity is needed. Polymerase chain reaction (PCR) is the most sensitive method for the detection of HBV DNA, but because of sample to sample variability in amplification efficiency, the quantification of target nucleic acids by conventional PCR is inaccurate. Therefore, we developed a competitive PCR method with an internal standard which uses the same primers as the target HBV DNA. The standard was generated from the HBV S gene by PCR site-directed mutagenesis and differed from the native S gene by a single base which introduced an internal restriction enzyme site. For practical application, this method was used to quantitate the effect of the carbocyclic analogue 2'-deoxyguanosine (2'-CDG) on HBV DNA replication in 2.2.15 cells, an HBV transfected HepG2 cell line. A decrease of HBV DNA level in the attomole range was demonstrated by the assay in the media of 2.2.15 cells treated with 2'-CDG. The results were verified by conventional HBV DNA slot blot analysis followed by densitometric scanning. Competitive PCR proved to be a sensitive, accurate and reliable method for HBV quantitation.
Wu
J
J
Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA.
Sullivan
D E
DE
Gerber
M A
MA
D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics* / Q000592:standards; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; C061610:2-amino-1,9-dihydro-9-(3-hydroxy-4-(hydroxymethyl)cyclopentyl)-6H-purine-6-one; D003849:Deoxyguanosine