应用突变特异PCR检测HBV前C区nt1896位点突变

Mutation specific PCR method for detecting nt1896 mutation in HBV pre-C region

目的建立检则HBV前C区突变的突变特异聚合酶链反应(msPCR)方法,探讨前C区突变与肝病临床分型、e系统状态及病毒复制的关系。方法根据HBV前C区nt1896G→A突变设计并合成引物,建立msPCR检测方法,通过测序和扩增产物电泳证实msPCR的特异性。结果229例急性肝炎、慢性肝炎轻度、中度、重度及肝硬化患者nt1896突变株感染率分别为25.0%、5.3%、5.2%、27.8%和20.0%;野生株和突变株混合感染率分别是62.5%、32.9%、50.6%、38.9%和38.0%。221例慢性HBV感染者中,野生株、突变株和混合感染者血清HBeAg阳性率分别为71.3%、34.8%和51.1%;HBVDNA含量分别为7.1×108±3.3×106、7.2×107±1.3×106和2.1×109±2.4×106copies/ml;ALT异常率分别为46.3%、73.9%和54.4%,突变组显著高于野生组(P<0.05)。干扰素治疗后,混合感染者血清HBVDNA的阴转率显著高于野生株(P<0.05)。结论msPCR检测nt1896突变灵敏特异,nt1896突变与肝病严重性有密切关系。

Objective To establish mutation specific polymerase chain reaction （msPCR） method for detecting pre-C mutation and to explore the relationship between pre-C mutation and clinical types,serum e system status and virus replication. Methods According to nt1896 G→A mutation, the primers were designed and msPCR was established. The specify of the msPCR was demonstrated by sequencing and electrophoresis of PCR products. Results Mutation and wild strain infections in 229 patients with HBV infection were detected using this msPCR method. The rate of nt1896 mutation infection in patients with acute hepatitis, light, moderate, severe chronic hepatitis and liver cirrhosis was 25.0%, 5.3%, 5.2%, 27.8% and 20.0%, respectively. The rate of mixed infection was 62.5%,32.9%, 50.6%, 38.9% and 38.0%, respectively. Among 221 patients with chronic HBV infection,the positive rate of HBeAg in wild, mutation and mixed infection was 71.3%, 34.8% and 51.1%,wild strain was significantly higher than the other groups; HBV DNA level was 7.1 × 10^8 ± 3.3 × 10^6,7.2 × 10^7 ± 1.3×10^6 and 2.1 × 10^9±2.4 × 10^6 copies/ml; the abnormal rate of ALT was 46.3%,73.9% and 54.4%. The mutation infection was significantly higher than the wild infection（P〈0.05）. After using 1FN treatment, the negative rate of serum HBV DNA in mixed infection was significantly higher than that in wild strain infection （P 〈 0.05）. Conclusions The msPCR method for detecting nt1896 G→A mutation was specific. Mutation of nt1896 G→A can inhibit synthesis and secretion of HBeAg and is associated with the severity of liver diseases.