鸭乙型肝炎病毒cccDNA Taq Man荧光定量PCR的建立

Establishment of the method for detecting DHBV cccDNA by the TaqMan fluorescence quantitative PCR

目的建立鸭乙型肝炎病毒(DHBV)cccDNA TaqMan荧光定量PCR方法。方法用pUCm-T载体和PCR纯化产物连接,转染TOP10菌,筛选阳性菌落,提取质粒,制作外标准品;在DHBV基因组负链两侧设计一对引物,并在引物间设计和荧光标记一段TaqMan探针,严格优化反应物的组成和扩增条件,建立了DHBV cccDNA TaqMan荧光定量PCR方法。结果最低可检出10~4拷贝/ml;批内误差为0.2%～3.14%,批间误差为2.22%～4.43%;在10~5～10^(12)拷贝/ml之间与C_t值具有很好的线性[Ct=-2.8361 ln(x)+41.45,r=-0.9985]。结论该法是一种灵敏度高、特异性强、较为简便的DHBV cccDNA定量检测技术。

Objeelive To develop a fluorescence quantitative PCR based on TaqMan chemistry for quantification of duck hepatitis virus B（DHBV） covalently closed circular DNA（cccDNA）. Methods The PCR fragment of DHBV DNA was cloned into vector pUCm-T. The recombinant plasmid was purified and subsequently quantified as HBV DNA standard. A pair of primers were designed from the two sides of the nick in the minus strand of DHBV-DNA and a TaqMan probe between the primers, modified with 6-FAM at 5＇-end and TAMP, at its 3＇-end, was designed to detect the PCR production during the PCR cycles. The experimental conditions and reagents of amplification were sophisticatedly optimized in order to produce perfect amplification efficiency and reduce non-specific amplification. Results The detectable limit of the assay was 104 copies/ml. A linear standard curve was obtained between 10s to 1012 copies/ml（Ct =-2.8361In（x） ＋41.45, r =-0.9985）. The coefficient of variation was 0.2% to 3.14% and 2.22% to 4.43% for intra-and inter-assay, respectively. After a dynamic survey of the DHBV DNA content in the ducks serum, we found its peak value appeared in the second week after the birth. Conclusion The TaqMan FQ-PCR for quantification of DHBV cccDNA is a simple, highly sensitive and specific method.