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目的建立一种肝活检组织中 HBV 共价闭合环状 DNA(cccDNA)的定量检测方法。方法待检肝组织标本共21份,来源于江苏省人民医院肝脏手术患者,包括19份慢性 HBV 感染,其中 HBeAg(+)标本10份,HBeAg(-)标本9份,4份非 HBV 感染为阴性对照组,取 HBV DNA 阳性的患者外周血作为 rcDNA 组。检测方法的主要步骤为肝组织经蛋白酶 K 和细胞裂解缓冲液消化后,用液相抽提法提取核酸,将提取的核酸溶液分为2等份。1份用特异性降解单链 DNA 的核酸酶加以消化,纯化后使用跨缺口引物和 SYBR Green Ⅰ荧光染料进行实时荧光定量 PCR 分析;另1等份则用以-定量检测肝细胞看家基因(β-Globin)作为样本细胞参数。检测结果的特异性主要通过 PCR 反应产物的序列分析及 rcDNA 组结果的对照进行证实,HBeAg(+)组和 HBeAg(-)组 cccDNA 水平的差异通过两样本 t 检验进行分析。结果 PCR 产物经琼脂糖凝胶电泳分析显示扩增产物的碱基数为350 bp左右,DNA 测序分析提示产物与目的片段的序列符合率为99%以上,且以 rcDNA 为对照的结果均为阴性,排除最有可能造成假阳性的 rcDNA 对结果的干扰。本方法对10 mg HBeAg(+)肝组织标本的cccDNA 的检测阳性率为100%。血清 HBeAg(+)的肝组织样本的平均 HBV cccDNA 水平高于HBeAb(+)的肝组织标本(P<0.05)。结论通过上述三种途径证实了本文所建立方法的特异性。应用 SYBR Green Ⅰ荧光染料和β-Globin 作为样本细胞参数所建立的实时荧光定量 PCR 方法检测肝细胞内 HBV cccDNA,具有较高的特异性、敏感性,且成本较低的特点。
Objective To establish a quantitative detection method of HBV covalently closed circular DNA (cccDNA) in liver biopsy tissues. Methods A total of 21 liver tissue samples were obtained from patients undergoing liver surgery in Jiangsu Provincial People’s Hospital, including 19 chronic HBV infections, including 10 HBeAg (+) specimens, 9 HBeAg (-) specimens and 4 non-HBV infection Negative control group, taking HBV DNA-positive peripheral blood as rcDNA group. Detection of the main steps of the liver tissue proteinase K and cell lysis buffer digestion, nucleic acid extraction by liquid phase extraction, the extracted nucleic acid solution is divided into two equal parts. One was digested with a nuclease that specifically degraded single-stranded DNA and purified by real-time fluorescent quantitative PCR analysis using a gap primer and SYBR Green I fluorescent dye. The other 1 aliquot was used to quantify hepatocellular housekeeping genes β-Globin) as sample cell parameters. The specificity of the test results was confirmed by sequence analysis of the PCR reaction products and the control of the rcDNA group results. The differences of cccDNA levels between the HBeAg (+) group and the HBeAg (-) group were analyzed by two-sample t-test. Results PCR products were analyzed by agarose gel electrophoresis showed that the number of base amplified products was about 350 bp, DNA sequencing analysis showed that the sequence of the product and the target fragment was 99% or more, and the results were negative for rcDNA , Excluding the most likely cause of false positive rcDNA interference on the results. The method for the detection of cccDNA in 10 mg HBeAg (+) liver tissue specimens was 100% positive. The average level of HBV cccDNA in serum of HBeAg (+) liver tissue samples was higher than that of HBeAb (+) liver tissue samples (P <0.05). Conclusions The specificity of the method established in this article is confirmed by the above three ways. The detection of HBV cccDNA in hepatocytes by real-time fluorescence quantitative PCR with SYBR Green Ⅰ fluorescent dye and β-Globin as the sample cell parameters is characterized by high specificity, sensitivity and low cost.