鸭肝组织中DHBV cccDNA的实时荧光定量PCR方法的建立

Development and Validation of Quantitative Duck Hepatitis B Virus cccDNA PCR Assays in Duck Liver Tissue

目的 建立基于DHBV病毒正、负链缺口区设计引物、沉淀蛋白结合的DNA、酶切消化残留线性DNA的DHBVcccDNA荧光定量PCR方法.方法 根据DHBV cccDNA和rcDNA结构的不同,将引物设计在DHBV DNA负链缺口的两侧.采用十二烷基硫酸钾蛋白质沉淀法分离肝组织中cccDNA和rcDNA.并对提取的DNA进行紫外定量,按照每2UPSAD消化500ng cccDNA,计算PSAD的用量,酶切消化残存的线性DNA.以pBR322/2DHBV Core重组质粒为PCR标准品,通过优化反应体系和扩增条件,建立检测DHBV cccDNA的SYBR Green I实时荧光定量PCR方法,并进行方法学考察和实际应用.结果 优化的PCR扩增产物电泳后可见239 bp的DNA片段,与目标片段长度相同.标准曲线回归方程Y=-4.085 7X＋48.805,R2=-0.997 6.方法的灵敏度为103 copies/ml,线性范围可达103～108 copies/ml.方法特异性强,未检出DHV,HBV及E coli DNA.对已感染DHBV的鸭肝组织检测DHBV cccDNA,进行定量,含量从0.36～1733.08 copies/diploid genome.其中分布最广的是10～99 copies/dioploid genome.DHBV cccDNA占总DHBV DNA的比例平均仅为3.86％,范围从0.01％～13.3s％.结论 根据DHBV cccDNA与rcDNA的结构和理化性质的差异,设计能够特异性扩增DHBV cccDNA的荧光定量PCR方法,方法灵敏度高、特异度强,可广泛应用于DHBV和HBV抗病毒治疗策略的相关研究中.

Objective To develop a Taqman real time fluorescent quantitative PCR assay for duck hepatitis B virus （DHBV） cccDNA,based on the gap region of positive and negative strand of DHBV DNA and prepared the samples by the precipitati on of protein binding DNA and the digestion of linear DNA.Methods According to the structure of DHBV cccDNA and rcDNA,the primers were designed to locate at the sides of the gap of negative strand of DHBV DNA.After the isolation of DHBV cccDNA and rcDNA from liver tissue by protein precipitation used dodecyl sulfate,linear DNA was digested by en zyme according to the digestion of 500 ng of cccDNA by 2U PSAD.The recombinant plasmid of pBR322/2DHBV Core gene was used to generate DHBV DNA standard.By optimizing the reaction system and amplification conditions,the SYBR Green I real time fluorescent quantitative PCR assay for DHBV cccDNA was developed,and methodological study was conducted.Results Optimized PCR amplification product was showed 239bp fragment by electrophoresis,which was the same length as the target fragment.The assay had a lowest detection limit of 103 copies/ml and a good linear standard curve （Y=-4.085 7X＋48.805,R2=0.997 6） over a wide range of DHBV cccDNA levels （103 to 108 copies/ml）.The specificity of the assaywas demonstrated by detecting duck hepatitis virus,HBV and E.coli DNA.Investigation of DHBV cccDNA of liver tissue from ducks infected by DHBV by the assay showed the content was from 0.36 to 1 733.08 copies/diploid genome,and the most widely distribution was 10～99 copies/dioploid genome.The proportion of DHBV cccDNA to total DHBV DNA was only 3.86％ on average,ranging from 0.01％ to 13.38％.Conclusion The FQPCR developed is highly sensitive and specif ic,and can be widely used in the research of anti DHBV or HBV treatment strategies.