不同核酸提取方法在HBV DNA荧光定量检测中的比较  

Comparison of the influence of different nucleic acid extraction methods on HBV DNA detection using quantitative fluorescence-PCR

作　　者：李成德[1] 黄晓佳[1] 陈雄毅[1] 

机构地区：[1]广东省肇庆市第一人民医院检验科,526021

出　　处：《国际检验医学杂志》2010年第12期1361-1363,共3页International Journal of Laboratory Medicine

摘　　要：目的研究不同核酸提取方法在HBVDNA荧光定量检测中的提取效率、重复性、抗干扰性及对扩增试剂的兼容能力。方法运用两种具有不同启动温度的Taq酶的扩增试剂分别扩增经一步法、煮沸法和磁珠法提取HBVDNA强阳性血清的模板,比较不同提取方法对扩增试剂的兼容能力。采用3种方法提取3份HBVDNA含量不同的标本各10次,进行扩增,比较不同方法的提取效率和重复性;对HBVDNA阳性标本用HBVDNA阴性的黄疸血清和溶血液进行10倍稀释后分别用前述方法提取进行干扰试验。结果 3种提取方法中,磁珠法和煮沸法提取的模板对两种扩增试剂具有良好的兼容性;重复性比较以一步法最佳,磁珠法次之,煮沸法较差;提取效率比较以磁珠法最理想,一步法次之,煮沸法最差;对黄疸和溶血的抗干扰能力以磁珠法最佳,溶血对煮沸法有一定影响,在无5-羧基-X-罗丹明(5-ROX)校正的情况下,黄疸对一步法影响较大,有5-ROX校正系统分析则可以消除这种影响。结论磁珠法具有良好的重复性和提取效率,对黄疸和溶血有较强抗干扰能力,对扩增试剂的兼容性好,是一种理想的核酸提取方法;相对而言,一步法快捷且重复性更佳。Objective To investigate the extraction efficiency, repeatability, anti interference ability and compatibility with amplification reagents of different nucleic acid extraction methods on HBV DNA detection by quantitative fluorescence PCR. Methods In order to compare the compatibility of different extraction methods with two kinds of amplification reagents containing various Taq DNA polymerases with different priming temperature, were used to amplify templates extracted respectively by one-step process,boiling method and magnetic beads method from a serum specimen with high HBV virus load. The three extraction meth- ods, mentioned above, were applied to prepare templates from three sample solutions, having different HBV virus load. Template of each solution was extracted for 10 duplicates by every method. Then the resulting nucleic acids were amplified to compare the extraction efficiency and repeatability of the three different methods. HBV DNA positive samples were diluted 10 times with HBV DNA negative serum from jaundice patient and with HBV DNA negative hemolysate serum,and then extracted by the three methods to test the interference effects from these specimens. Results Of the three extraction methods,templates extracted by magnetic beads method or boiling method had good compatibility with two amplification reagents. One-step method had the most high repeat- ability among the three methods and boiling method was the poorest. For extraction efficiency, magnetic beads method was the best, and the boiling method was the worst. As to anti interference ability for jaundice and hemolysis specimen, magnetic beads method was the best and no interference was detected. Interference effect was observed for boiling method within hemolysate specimen. Strong interference was detected for one step method within jaundice specimen, when there was no ROX used for correction, but this interference could be eliminated completely by using ROX as reference dye. Conclusion Magnetic beads method is an ideal method for nucleic acid

关 键 词：核酸扩增技术 肝炎病毒 乙型 聚合酶链反应一步法 煮沸法 磁珠法 

分 类 号：R440[医药卫生—诊断学]