Simultaneous detection of HBV-DNA concentration and its melting temperature by improved fluorescent quantitative PCR

Objectives To establish sensitive and specific assay of simultaneous detection of HBV-DNA concentration and its melting temperature(Tm) by improving fluorescent quantitative polymerase chain reaction(FQ-PCR) of single label fluorescence. Methods Three cases with HBV-DNA positive serum(108.48,105.70 and 103.70copies/ml ) and 1 case HBV-DNA negative serum were tested by the TaqMan porbe and SYBR Green I dye double label FQ-PCR (T+S group), SYBR Green I dye single label FQ-PCR(S group) and TaqMan probe single label FQ-PCR(T goup). PCR cycling and DNA melting parameters were set up to detect HBV-DNA concentration and its Tm,and each serum was tested 5 times by PCR assay. Results The HBV-DNA positive sera were 100% in both T+S group and T group.Their HBV-DNA concentrations were 108.55±0.32,105.79±0.29,103.81±0.30 and 108.49±0.31,105.69±0.30,103.72±0.25 copies/ml respectively.They have no statistically significant difference(t=0.31,0.54 and 0.27 resectively, P0.05). The HBV-DNA positive rate was 86.7% in S group, their HBV-DNA concentrations were 108.41±0.35,105.21±0.34 and 103.26±0.26 copies/ml, and the detected concentrations were lower than those of the T+S group (t=2.90,2.62 respectively, P0.05),except for the highest HBV-DNA concentration.The melting curve of HBV-DNA was detected in both T+S group and S group,and each ordinal Tm was 79.8℃,71.8℃ and 72℃ respectively.Sensitivity of FQ-PCR testing HBV-DNA in T+S group, T goup and S group were 102.70,102.70 and 103.00 copies/ml respectively.Conclusions Sensitivity and specificity of testing HBV-DNA by improving FQ-PCR of double fluorescent label of TaqMan probe and SYBR Green I dye are higher,and the improving FQ-PCR can be applied to test HBV-DNA concentration and its Tm simutaneously,which provides new idea for testing HBV-DNA and its polymorphism,especially identification of HBV genetype.