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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: To rapidly extract HBV DNA from ascites cells with cytolytic lysate and to establish a real-time fluorescence quantitative PCR for the detection of HBV DNA, and to study its clinical significance. Methods: The ascites of patients with hepatitis B cirrhosis were centrifuged at 10 ml for 1 min and 1500 g / ml for 5 minutes. The supernatant was aspirated and the fluorescence quantitative detection of HBVDNA in the sediment cells was performed. Results: Fluorescent PCR was used to detect HBV DNA in ascites cells, and the detection value reached 102 IU / ml. The dilution ratio showed a theoretical gradient with a correlation coefficient of 0.91. This method has good sensitivity. Repeated studies found that CV values of 5 replicates were 14.4%, 9.8% and 11.2%, respectively, in line with the deviation of the Ministry of Health Clinical Testing Center requirements. The content of HBV DNA in ascites cells was significantly higher than that in ascites, the difference between the two was 0.5 ~ 1.5 orders of magnitude, indicating that ascites cells contain not only a large number of HBV, but also significant differences between individuals. A case of hepatitis B hepatocellular carcinoma patients with ascites three times every 5 days for sediment cells HBV DNA dynamic detection and found that the sediment cells of HBV DNA and serum HBV DNA at the same time with the patient’s condition was gradually decreased. Conclusion: The determination of HBV DNA in ascitic cells based on the detection of fluorescent PcR is simple, sensitive and reproducible, which can be used as a means to study the clinical significance of intracellular HBV DNA in ascites.