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目的:采用细胞核酸释放剂(Cell nucleic releaser,CNR),建立在同一PCR扩增管内同时进行细胞核酸提取和荧光PCR扩增的白细胞HBV DNA检测技术。方法:白细胞的处理:新鲜抗凝(EDTA-Na2)全血0.2 ml,破碎红细胞,用生理盐水洗涤四次,弃净残液,加20μl白细胞温化剂制备成白细胞悬液。白细胞HBV DNA核酸提取与扩增:取3μl的CNR和等量的白细胞悬液在PCR扩增管底轻轻吹打混匀,覆盖30μl无菌石蜡油,37℃,60 s→99℃,7 min→37℃,30 s,直接加入已配制荧光PCR反应液,3000 g/min离心1 min,进行实时荧光定量PCR扩增。采用QiaGen的核酸提取方法作为对照。选择144例血清HBV DNA检测结果大于104IU/ml和131例血清无HBV DNA检出的乙肝患者标本为研究对象,20例非乙肝患者全白细胞作为特异性研究标本。结果:采用融红细胞的方法分离白细胞和CNR分离核酸,无白细胞和核酸丢失之嫌,可对全白细胞内的HBV DNA进行准确定量,该方法具有较好的灵敏性和重复性。与QiaGen核酸提取方法相关性良好(r=0.96),144例血清HBV DNA大于104IU/ml的乙肝患者其白细胞全部大于100 IU/ml,131例血清HBV DNA阴性的乙肝患者有56例(42.7%)白细胞HBV DNA大于100 IU/ml。结论:基于CNR核酸提取方法的全白细胞HBV DNA定量技术,操作简单、且具有较好的敏感性和重复性,可作为研究白细胞内HBV DNA临床意义的检测手段。
OBJECTIVE: To establish a method for detection of leucocyte HBV DNA by simultaneous cell nucleic acid extraction and fluorescent PCR amplification in the same PCR amplification tube by using cell nucleic acid releaser (CNR). Methods: Treatment of leukocytes: 0.2 ml of fresh whole anticoagulant (EDTA-Na2) whole blood, crushed red blood cells, washed four times with saline, abandoned net residue, add 20 μl of leukocyte warming agent to prepare a white blood cell suspension. Leukocyte HBV DNA Nucleic Acid Extraction and Amplification: Take 3μl of CNR and the same amount of leukocyte suspension and gently mix with 30μl of sterile paraffin oil at 37 ℃, 60 s → 99 ℃ for 7 min → 37 ℃, 30 s, directly added to the fluorescence PCR reaction solution was prepared, 3000 g / min centrifugation 1 min, real-time quantitative PCR amplification. A QiaGen nucleic acid extraction method was used as a control. A total of 144 cases of HBV DNA test results greater than 104IU / ml and 131 cases of serum HBV DNA were detected in patients with hepatitis B specimens as the research object, 20 cases of non-hepatitis B patients with leukocytes as a specific study specimens. Results: The leucocyte and CNR isolated from leucocytes were isolated from leucocytes and CNR without any leucocyte and nucleic acid loss, which could accurately quantify HBV DNA in leukocytes. This method has good sensitivity and repeatability. There were 56 leukocytes (100%) in all HBV patients with HBV DNA greater than 104 IU / ml in 144 patients (42.7%) with 131 HBV DNA-negative hepatitis B patients (r = 0.96) ) Leukocyte HBV DNA is greater than 100 IU / ml. Conclusion: Based on CNR nucleic acid extraction method, HBV DNA quantification technique of whole leukocytes is easy to operate and has good sensitivity and repeatability. It can be used as a detection method for clinical significance of HBV DNA in leucocytes.