Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium.
Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.
Pan
Xiao-Ben
XB
Peking University Hepatology Institute, Peking University People's Hospital, Beijing, PR China.
Wei
Lai
L
Han
Jin-Chao
JC
Gao
Yan
Y
D002460:Cell Line; D002877:Chromosomes, Human / Q000737:chemistry* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis*; D005453:Fluorescence; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D003470:Culture Media; D004279:DNA, Viral