PCR介导的重组影响乙型肝炎病毒共价闭合环状DNA的分析。

StephenW

Retrovirology. 2016 Dec 20;13(1):84. doi: 10.1186/s12977-016-0318-1.
PCR mediated recombination impacts the analysis of hepatitis B Virus covalently closed circular DNA.Suspène R1, Thiers V1, Vartanian JP2, Wain-Hobson S3.
Author information

• 1Molecular Retrovirology Unit, Institut Pasteur, 28 rue du Dr. Roux, 75724, Paris Cedex 15, France.
• 2Molecular Retrovirology Unit, Institut Pasteur, 28 rue du Dr. Roux, 75724, Paris Cedex 15, France. [email protected].
• 3Molecular Retrovirology Unit, Institut Pasteur, 28 rue du Dr. Roux, 75724, Paris Cedex 15, France. [email protected].
  

AbstractBACKGROUND: The replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination.
RESULTS: The impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination.
CONCLUSIONS: PCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.


KEYWORDS: HBV; PCR recombination; cccDNA; rcDNA

StephenW

逆转录病毒。 2016 Dec 20; 13（1）：84.doi：10.1186 / s12977-016-0318-1。
PCR介导的重组影响乙型肝炎病毒共价闭合环状DNA的分析。
SuspèneR1，Thiers V1，Vartanian JP2，Wain-Hobson S3。
作者信息

1分子逆转录病毒学单位，Institut Pasteur，28 rue du Dr.Roux，75724，Paris Cedex 15，France。
2 Molecules Retrovirology Unit，Institut Pasteur，28 rue du Dr.Roux，75724，Paris Cedex 15，France。 [email protected]。
3分子逆转录病毒学单位，Institut Pasteur，28 rue du Dr.Roux，75724，Paris Cedex 15，France。 [email protected]。

抽象
背景：

HBV的复制涉及通过修复病毒颗粒中易于环化的DNA（rcDNA）来从HBV基因组产生共价闭合的环状DNA（cccDNA）。由于cccDNA是HBV基因组的转录模板，因此需要从PCR定量中去除cccDNA拷贝数，这是评价治疗方案效率的首选技术。通常用于鉴定cccDNA的PCR靶标跨越rcDNA的缺口区域，并且被认为是有一个潜在的混杂问题，即PCR可以从小DNA片段的集合产生更大的靶标，这种现象被称为PCR重组。
结果：

通过混合三个标记的，但重叠的HBV DNA片段来研究PCR重组对该cccDNA特异性靶标的扩增的影响。通过用范围从0.6至23％的频率测序鉴定十六种可能的重组体。为了在体内证实这一发现，用DNA酶I处理HBV阳性血清，并提交定量实时PCR。在这些条件下，可以毫无困难地扩增cccDNA特异性区段。由于病毒体包含独特的rcDNA，来自PCR重组的cccDNA靶的扩增。
结论：

cccDNA的PCR定量可能比以前认为更困难。当前的检测方案需要进行调查，以帮助管理慢性HBV感染。
关键词：

HBV; PCR重组; cccDNA; rcDNA