Guo Yongcan, Sheng Shangchun, Nie Bin, Tu Zhiguang
The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China.
Hepat Mon. 2015 Jan 5;15(1):e23729. doi: 10.5812/hepatmon.23729. eCollection 2015 Jan.
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available.
Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology.
The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B.
Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 10(2)-10(6) IU/mL was revealed (r(2) = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA.
The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.
乙型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）在病毒生命周期和持续感染中起着至关重要的作用。然而，尚无用于cccDNA检测的特异性和定量方法。
我们旨在开发并初步评估一种基于磁捕获杂交和定量PCR技术的HBV cccDNA定量方法。
设计了特异性捕获位于松弛环状DNA（rcDNA）缺口两侧的HBV cccDNA的功能化纳米颗粒。然后，开发了磁捕获杂交和定量PCR（MCH-qPCR）检测方法，并使用cccDNA标准品和慢性乙型肝炎患者的血清样本对其性能进行了初步评估。
制备了特异性的cccDNA捕获纳米颗粒，成功开发了一种用于cccDNA的磁捕获杂交和定量检测方法。检测限为90 IU/mL，MCH-qPCR在10(2)-10(6) IU/mL范围内显示出良好的线性关系（r(2) = 0.994）。与直接实时PCR相比，高含量的HBV DNA不影响MCH-qPCR方法对cccDNA的检测，并且cccDNA与rcDNA之间没有交叉反应。
新型MCH-qPCR方法具有良好的敏感性和特异性。它可以满足临床常规检测的要求。
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