摘要
建立检测血清HBV DNA的多聚酶链反应技术。人工合成adr、adw和ayw三个业型HBV的两个共有序列NC1和NC2作为引物扩增一长428bp且含一BglⅡ酶切点的HBV C基因片段。含FD酶的反应体系在水浴93℃30秒、55℃60秒、72℃90秒依序循环30周期,当克隆HBV DNA模板最小加量为10 fg时,产物作琼脂糖凝胶电泳经溴化乙锭染色在紫外线下观察仍可见428 bp位置的阳性荧光带。阳性产物经BgⅠⅡ酶切后分为125 bp和303 bp的两个特异片段。同一条件下的阴性对照则出现阴性结果。这一技术用以检测经常规DNA提取的127例HBsAg阳性血清,结果HBV DNA检出率达88％,其中HBeAg阳性血清HBV DNA检出率为97％,抗HBeAg阳性血清HBV DNA检出率为82％。
The method of polymerase chain reaction (PCR) for detection of hepatitis B virus (HBV) DNA in serum was established. Two primers of 20 nucleotides each , NC1 (1955-1974) and NC2(2287-2268 R), co-conserved by subtype adr, adw and ayw, were synthesized. HBV DNA of 428 bp containning a restriction site of Bgl Ⅱ is to be amplified, which cut it into two, one 125 bp, another 303 bp. With FD DNA polymerase and 30 yccles, each in water bath in 93℃ for 30 seconds, 55℃ for 60 seconds, 72℃ for 90 seconds and finally in 72℃ for 10 minutes, the specific product of the reaction and its restrictive fragments were determined in agarose gel electrophoresis under ultraviolet light when 10 fg HBV DNA was used for template. With the technique, 127 HBsAg positive sera were tested and HBV DNA was pick up in 88%, among them HBV DNA was detected in 97% of HBeAg positive sera and 82% of anti-HBe positive sera.
出处
《中山医科大学学报》
CSCD
1992年第3期44-47,共4页
Academic Journal of Sun Yat-sen University of Medical Sciences
关键词
聚合酶链反应
乙型肝炎病毒
DNA
polymerase chain reaction (PCR)
hepatitis B virus (HBV)
deoxyribo-nucleic acid (DNA)
restriction enzyme
