乙型肝炎病毒共价闭合环状DNA套式实时荧光定量聚合酶链反应的建立

Establishment of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

目的建立检测HBV共价闭合环状DNA（ceeDNA）的套式-实时荧光定量PCR法。方法根据HBVeecDNA与松环DNA（rcDNA）结构上的差异，设计2对跨缺口的特异引物及1条位于负链缺121下游的特异TaqMan荧光探针。根据Plasmid—Safe^TM ATP—Dependent DNase（PSAD）对rcDNA与cceDNA作用的不同，对模板DNA进行酶切纯化，降解rcDNA，再进行套式PCR扩增，先用外引物和模板进行第一轮常规PCR，再用内引物、荧光探针和第一轮PCR产物进行实时荧光定量PCR，根据阳性参照标准品，得出待检标本定量值。结果检测阳性参照标准品，得出该方法灵敏度可达21g拷贝／mL。用上述方法检测34份乙型肝炎患者血清HBV DNA阳性标本，25份血清HBV eccDNA阳性，28份外周血单个核细胞HBV cceDNA阳性。27份健康对照者血清HBV DNA阴性标本，6份HBV cecDNA阳性。对5份HBV cccDNA阳性标本扩增产物进行克隆测序，无碱基缺失、突变。与HBV不同基因型序列（A～G）比较，同源性为90．6％～99．1％，其中，与B、C基因型同源性为95．3％～99．1％，验证了方法的特异度。结论套式一实时定量PCR法可检测乙型肝炎患者血清、PBMC中的HBV cccDNA，且具有敏感、特异性。

Objective To establish a nested real-time quantitative polymerase chain reaction （PCR） assay for detection of hepatitis B virus covalently closed circular DNA （HBV cccDNA）. Methods Based on the sequence differences between HBV cccDNA and hepatitis B virus relaxed circular DNA （ HBV rcDNA）, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream of the negative chain were designed. To remove rcDNA, sample DNA was processed by Plasmid-SafeTM ATP-Dependent DNase, and then amplificated by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. On the basis of the copy number of positive standard preparation, DNA levels of specimen were defined. Results Sensitivity of nested real-time quantitative PCR was 2 lg copy/mL based on positive standard preparation. Then 34 samples of the sera from patients with chronic hepatitis B （CHB） and 27 from healthy controls were tested. Among 34 patients with serum HBV DNA positive, 25 were serum HBV cccDNA positive, 28 were HBV cccDNA positive in peripheral blood mononuclear cells. Among 27 healthy controls with. serum HBV DNA negative, 6 were serum HBV cccDNA positire. Five samples with HBV cccDNA positive were cloned and sequenced. And no base deletion and mutation was detected. Compared with different HBV genotypes （A-- G）, the homology was 90.6 % --99. 1%, and it was 95.3% --99. 1% compared with genotype B and C, which showed a high specificity. Conclusions The nested real-time quantitative PCR possesses high specificity and sensitivity. It may be applied to detect HBV cccDNA in serum and PBMC of CHB patients.