Detection of HBV-DNA by the Double Labeled Fluorescent Quantitative Polymerase Chain Reaction

Objective: To explore the significance of testing HBV-DNA by double labeled fluorescent quantitative polymerase chain reaction of SYBR Green Ⅰ and TaqMan probe(dFQ-PCR).Methods: Four samples at different HBV-DNA serum levels(1011.70,108.70,106.70 and1×106.00 copies/L) were tested by dFQ-PCR.Each of the results was compared with that assayed by SYBR Green Ⅰ and TaqMan probe single labeled FQ-PCR(sFQ-PCR) respectively.PCR cycling and DNA melting parameters were setup to detect HBV-DNA concentration and the melting time(Tm),and every sample were tested 5 times by every PCR assay.Results: All HBV-DNA positive serum were detected by dFQ-PCR and TaqMan labeled sFQ-PCR,their HBV-DNA concentration were 1011.55±0.32,108.79±0.29,106.81±0.30 and 1011.49±0.31,108.69±0.30,106.72±0.25 copies/L respectively,without statistically difference(t=0.31,0.54 and 0.27,P0.05 respectively).HBV-DNA concentration detected by SYBR Green Ⅰ labeled sFQ-PCR were 1011.41±0.35,108.21±0.34 and 106.26±0.26(only in HBV-DNA positive serum) copies/L.It was lower than that of the dFQ-PCR at low and medium concentration(t=2.90 and 2.62,P0.05,respectively).The melting curve were detected in both dFQ-PCR and SYBR Green Ⅰ labeled sFQ-PCR,their Tm were 79.8,71.8 and 72 ℃ respectively.Conclusion: Simultaneity dFQ-PCR can be applied to test HBV-DNA concentration and Tm,which provides a new testing method for HBV-DNA level and HBV genotype detection.