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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％。
To establish a polymerase chain reaction for the detection of serum HBV DNA. Two common sequences NC1 and NC2 of three types of HBV, adr, adw and ayw, were synthesized and used as primers to amplify a 428 bp HBV C gene fragment containing a Bgl Ⅱ cleavage site. The reaction system containing FD enzyme was cycled for 30 cycles in a water bath at 93 ° C for 30 seconds, at 55 ° C for 60 seconds and at 72 ° C for 90 seconds. When the minimum amount of cloned HBV DNA template was 10 fg, the product was subjected to agarose gel electrophoresis Ethidium bromide staining is still visible in the UV 428 bp position of the positive fluorescent bands. The positive product was digested with BgⅠⅡ into two specific fragments of 125 bp and 303 bp. Negative controls under the same conditions showed negative results. This technique was used to detect 127 cases of HBsAg-positive serum extracted by conventional DNA. The detection rate of HBV DNA was 88%. The detection rate of HBeAg-positive serum HBV DNA was 97%, and the detection rate of anti-HBeAg-positive serum HBV DNA 82%.