双荧光标记定量PCR检测乙型肝炎病毒核酸  

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

作　　者：黎炜英[1] 赵友云[2] 高应林[2] 王春香[2] 乐惠荣[2] 

机构地区：[1]武汉大学期刊社,湖北武汉430072 [2]湖北省中医院检验科,湖北武汉430061

出　　处：《武汉大学学报（医学版）》2007年第4期529-531,共3页Medical Journal of Wuhan University

摘　　要：目的:探索SYBR GreenⅠ和TaqMan探针双标记荧光定量PCR(dFQ-PCR)检测乙型肝炎病毒核酸(HBV-DNA)的意义。方法:选择浓度为1011.70,108.70,106.70和<1×106.00copies/L的4种血清各1份,进行dFQ-PCR,同时以TaqMan探针和SYBR GreenⅠ单荧光标记PCR(sFQ-PCR)为对照,设置同一扩增和熔解曲线的循环参数,检测HBV-DNA含量及其熔解温度(Tm),每种方法一次检测每份血清5次。结果:dFQ-PCR检测的HBV-DNA阳性血的平均含量为1011.55±0.32,108.79±0.29,106.81±0.30,与TaqMan探针sFQ-PCR的1011.49±0.31,108.69±0.30,106.72±0.25copies/L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,均为P>0.05);与SYBRGreenⅠ染料sFQ-PCR的1011.41±0.35,108.21±0.34和106.26±0.26copies/L(不含未检出的两次血清)比较,中低浓度有统计学意义(t=2.90和2.62,P<0.05)。dFQ-PCR和SYBR GreenⅠ染料sFQ-PCR均出现明显熔解曲线,HBV-DNA含量为1011.70,108.70和106.70copies/L的Tm分别为79.8,71.8和72℃。结论:dFQ-PCR能同时检测HBV-DNA含量和Tm,为HBV含量及其基因分型的同步检测提供了新思路。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 （10^11.70, 10^8.70, 10^6.70 and〈1×10^6.00 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 （Tin）, 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 10^11.55±0.32 , 10^8.79±0.29 , 10^6.81±0.30 and 10^11.49±0.31 , 10^8.69±0.30 , 10^6.72±0.25 copies/L respectively, without statistically difference （t = 0. 31, 0. 54 and 0. 27, P〉 0. 05 respectively）. HBV-DNA concentration detected by SYBR GreenⅠlabeled sFQ-PCR were 10^11.41±0.35, 10^8.21±0.34 and 10^6.26±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, P〈0.05, 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.

关 键 词：SYBRGreenⅠ TAQMAN探针 定量PCR HBV-DNA 

分 类 号：R373.4[医药卫生—病原生物学]