Combination of SYBR Green I and TaqMan Probe in the Detection of HBV-DNA by Fluorescent Quantitative PCR

Objective To explore the improvement of detecting HBV-DNA by fluorescent quantitative polymerase chain reaction(FQ-PCR)using both SYBR green I and TaqMan probe in the reaction.Method 3 HBV-DNA positive serum samples(108.48,105.70 and 103.70 copies/ml)and 1 HBV-DNA negative serum sample were used to detect HBV-DNA by the double fluorescent quantitative PCR(TaqMan+SYBR Green I group),SYBR Green I PCR(SYBR Green I group)and TaqMan probe PCR(TaqMan goup).The detection of the HBV-DNA of each sample was performed for 5 times.Result The HBV-DNA concentrations of the 3 HBV-DNA positive serum samples detected by TaqMan+SYBR Green I PCR were 108.55±0.32,105.79±0.29 and 103.81±0.30,while those detected by TaqMan PCR were 108.49±0.31,105.69±0.30,103.72±0.25 copies/ml.There was no statistically significance between two methods(t=0.31,0.54 and 0.27,resectively P 0.05).The HBV-DNA concentration detected by SYBR Green I PCR of the 3 positive samples were 108.41±0.35,105.21±0.34 and 103.26±0.26 copies/ml.The sensitivity of SYBR Green PCR in detecting HBV-DNA showed no significant difference to that of TaqMan+SYBR Green I PCR for the sample with a high HBV-DNA copy number(108.48 copies/ml)(t=0.68,P0.05),but was significantly lower for the samples with lower HBV-DNA copy number(105.70 and 103.70 copies/ml)(t=2.90,2.62,respectively P0.05).The melting curves were detected in both TaqMan+SYBR Green I group and SYBR Green I group,with Tm of 71.8℃,72℃ and 79.8℃,but was not detected in HBV-DNA negative serum samples.Conclusion Sensitivity and specificity of detecting HBV-DNA by SYBR Green I combined with TaqMan PCR are higher than only using SYBR Green I or TaqMan probe.