腹水细胞HBV DNA荧光PCR检测方法的建立及意义研究

Studying and the significance of the HBV DNA from ascite cells with time fluoresceence quantitative PCR

目的：采用细胞病毒裂解液快速提取腹水细胞内的HBV DNA,建立实时荧光定量PCR检测HBV DNA的技术,并初步研究其临床意义。方法：采用乙肝肝硬化患者腹水10 m l,1500 g/m in离心5 m in,全部吸出上清,对沉渣细胞进行HBVDNA荧光定量检测。结果：采用荧光PCR技术检测腹水细胞HBV DNA,检测出的数值可达到到102 IU/m l,并且按稀释倍数呈理论梯度,相关系数为0.91,该方法具有良好的敏感性。进行重复性研究发现,5次重复的CV值分别为14.4%、9.8%和11.2%,符合卫生部临床检验中心的偏差要求。腹水细胞HBV DNA含量明显高于单纯腹水中的HBVDNA,二者之差为0.5~1.5个数量级,说明腹水细胞不但含有大量的HBV,而且个体差异明显。对一例乙肝肝癌并发腹水患者每间隔5 d进行三次沉渣细胞HBV DNA动态检测,发现沉渣细胞的HBV DNA与血清HBV DAN同时随患者病情加重而呈逐渐下降趋势。结论：基于用荧光PcR的检测方法来测定腹水细胞HBV DNA,操作简单,具有较好的敏感性和重复性,可作为研究腹水细胞内HBV DNA临床意义的检测手段。

Objective：The Cyto-megalovirus CMV Lysis Solution,which can extraction the HBV DNA from ascite cells,has been used in detecting HBV DNA with time fluoresceence quantitative PCR,and then we can study the change and the clinical significance.Methods：10 ml ascite from cirrhosis patients originating hepatitis B infection centrifuged immediately by 1500 g for 5 minutes.The liquid supernatant was discarded thoroughly.The HBV DNA in ascite cells from the bottom of tube is detected with real time PCR technology.Results：The detected numerical attain abilities was 102 IU/ml,which using fluorescence PCR technology testing ascite cell HBV DNA it showed theoretical gradient according to the terms of diluted times.and then the Correlation coefficient is 0.91,making it clear that the method has a good sensitivity.By making repeated study we find that the 5 repeated times leafs CV is 14.4%,9.8% and 11.2%,and the declination meet the NCCL Requirements.There is about 0.5 to 1.5 LOG of HBV DNA in different ascite cells from patients with cirrhosis,which explaining ascite cells not only contains a lot of HBV,but also has much individual differences apparently.The HBV DNA in three times of dynamic detecting in one case of hepatocellular carcinoma is gradually decreased both in ascite and ascite cells along with the deterioration of disease.Conclusion： Our real-time PCR system of HBV DNA detection from ascite cells by the Cyto-megalo virus CMV Lysis Solution,can provide an accurate and highly sensitive rapid method to quantify Hepatitis B virus with low artificial positive and lower negative artificial results.It is suitable for studying the significance of HBV DNA from ascite cells in clinic.