摘要
目的探索SYBR GreenI联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为108.48、105.70和103.70copies/ml的3种HBV-DNA阳性血清和<1×103.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green I组成双荧光PCR(TaqMan+SYBR Green I组),同时进行TaqMan和SYBR Green I的单荧光PCR(分别为TaqMan组和SYBR Green I组),设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan+SYBR Green I组检测的HBV-DNA阳性血清均为阳性,其平均含量为108.55±、105.79±、103.81±,与TaqMan组的108.49±、105.69±、103.72±copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义(t=0.31、0.54和0.27,P>0.05);与SYBR Green I组的108.41±、0.35105.21±和103.26±copies/ml(不含未检出的两次血清)比较,除高浓度外,中低浓度有统计学意义(t=2.90和0.340.262.62,P<0.05)。TaqMan+SYBR Green I组和SYBR Green I组阳性血清均出现明显熔解曲线,熔解温度(Tm)分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green I联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。
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 （10^8.48, 10^5.70 and 10^3.70 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 10^8.55±0.32, 10^5.79±0.29 and 10^3.81±0.30, while those detected by TaqMan PCR were 10^8.49±0.31, 10^5.69±0.30, 10^3.72±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 10^8.41±0.35, 10^5.21±0.34 and 10^3.26±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 （10^8.48 copies/ml） （t=0.68, P〉0.05）, but was significantly lower for the samples with lower HBV- DNA copy number （10^5.70 and 10^370 copies/ml） （t=2.90, 2.62, respectively P〈0.05） . 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.
出处
《热带医学杂志》
CAS
2007年第6期575-577,共3页
Journal of Tropical Medicine
