HBV cccDNA荧光定量PCR检测方法的建立及初步应用

Establishment and application of a fluorescent quantitative pcr method for cccDNA of HBV

目的:评估一个新设计的HBV cccDNA荧光定量PCR检测方法。方法:以肝穿组织为检测标本,采用LightcycleTM探针,通过一对特殊引物,使HBVcccDNA可以扩增而病毒基因组不能扩增;同时利用一种依赖于ATP的特异性DNase提高反应的特异性,以此实现对cccDNA的检测。结果:成功建立了HBV cccDNA荧光定量PCR的检测方法,线性范围为2.5×101～2.69×109拷贝/ml。对5个病例的肝穿样本进行分析,其中3例为乙肝感染患者,并均接受抗病毒治疗达1年,另2例为乙肝阴性患者。检测结果如下:1)2例乙肝阴性患者的肝组织检测结果cccDNA及tDNA皆为阴性。2)接受拉米夫定治疗1年的病人tDNA为7.10×103拷贝/ml,cccDNA为2.64×103拷贝/ml;接受恩替卡韦治疗1年的病人tDNA为1.00×105拷贝/ml,cc-cDNA为4.04×103拷贝/ml;第3例病人接受拉米夫定治疗前tDNA为6.62×103拷贝/ml,cccDNA为3.03×102拷贝/ml,拉米夫定治疗1年后tDNA为5.19×103拷贝/ml,cccDNA为1.51×102拷贝/ml。结论:本研究结果显示,所建立的HBVcccDNA荧光定量PCR方法具有良好的线性范围,灵敏度、特异性及准确性均达到预期目标,而且检测所需样本量少,只需约1mg肝穿组织。可作为临床监测抗病毒治疗效果的有效手段和开展HBV复制调控研究的重要工具。

Objective：To evaluated a new fluorescent quantitative PCR method for HBV cccDNA. Methods：The specimens were obtained by hepatic puncturation. We designed a pair of specific primers to amplify not HBV genome but cccDNA as a method to detect it, using the ATP dependent Dnase to increase the specificity greatly. Results：We have established a new fluorescent quantitative PCR method for HBV cccDNA successfully, the linear range is from 2.5 ×10^1 to 2.69 × 10^9copies per milliliter. The three of five patients were shown HBV positive and the other two were negative using the new method to analyze hepatic puncturation specimens. The HBV positive patients have all received one year long anti-virus therapy. 1） the two HBV negative patients were both cccDNA negative and tDNA negative. 2） the patient who has received one year long Lamivudine therapy had a DNA concentration of 7.10 × 10^3copies tDNA per milliliter serum and 2.64 × 10^3copies cccDNA per milliliter serum. The patient who has received one year long entecavir therapy had a DNA concentration of 1.0 × 10^5copies tDNA per milliliter serum and 4.04× 10^3 copies cccDNA per milliliter serum in his blood, tDNA concentration in the third patient was 6.62 × 10^3copies per milliliter serum,3.3 × 10^2copies cccDNA per milliliter serum before anti-virus therapy, but after one year long Lamivudine therapy, the tDNA and cccDNA concentration reduced to 5.19 × 10^3copies and 1.51 × 10^2copies per milliliter serum respectively. Conclusion：This study shown that the fluorescent quantitative PCR had a good linear range,high sensitivity,specificity and accuracy,so it needs less specimen,for example 100 milligram hepatic puncturation specimen is enough. Although further study is needed,the fluorescent quantitative PCR will definitely become an efficient tool for anti-virus therapeutic effect monitoring because of its high sensitivity, specificity,efficiency and accuracy.