乙型肝炎病毒DNA的实时PCR采用荧光杂交探针定量迅速

StephenW

Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes

    Authors: Stephen K.N. Ho1,  Wing-Cheong Yam1,  Eric T.K. Leung1,  Lei-Po Wong1,  Jack K.H. Leung1,  Kar-Neng Lai1, Tak-Mao Chan1
        
        Affiliations: 1 Departments of Medicine1 and Microbiology2, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China
    Correspondence Tak-Mao Chan [email protected]
    J. Med. Microbiol., May 2003 52: 397-402, doi: 10.1099/jmm.0.05071-0
    Subject: Diagnostics, Typing And Identification

    Received: 11/09/2002 Accepted: 20/01/2003 Published Online: 01/05/2003



    A highly sensitive and rapid assay has been developed to quantify hepatitis B virus (HBV) DNA, based on the fluorescence resonance energy transfer principle and real-time PCR, using the LightCycler and a pair of specific fluorescent hybridization probes. This LightCycler real-time PCR assay (LC-PCR) detected HBV DNA in a linear range from 10**1 to 10**8 copies per reaction (250–2.5 × 10**9 copies ml−1), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards (ad and ay subtypes) and exhibited low intra-assay (< 6 %) and inter-assay (< 16 %) variation for both subtypes over the complete range of 7 orders of magnitude. The assay was evaluated clinically using serum samples from 120 HBsAg+ individuals and 45 healthy controls who were negative for both HBsAg and anti-HBc. Levels of HBV DNA were measured in these samples using both the LC-PCR and Digene Hybrid Capture II HBV DNA (HCII) assays. The prevalence rates for HBV DNA in the HBsAg+ serum samples were respectively 95 % (114/120) and 56 % (67/120) by LC-PCR and HCII (P < 0.01). All 67 HCII-positive samples tested positive with LC-PCR, while the 47 discordant samples showed low levels of HBV DNA (down to 265 copies ml−1), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation (r = 0.902; P < 0.001). The level of HBV DNA was significantly higher in HBeAg+ than anti-HBe+ samples (median 1.5 × 107 vs 4.6 × 104 copies ml−1; P < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.

    Abbreviations: CV, coefficient of variation; FRET, fluorescence resonance energy transfer; HBV, hepatitis B virus; HCII, Digene Hybrid Capture II; LC-PCR, LightCycler PCR.

StephenW

乙型肝炎病毒DNA的实时PCR采用荧光杂交探针定量迅速

    作者：斯蒂芬·K.N. 110 1，永畅Yam1，埃里克T.K。 Leung1，雷宝Wong1，杰克K.H. Leung1，嘉NENG Lai1，德毛CHAN1
        
        兼职：1部Medicine1和Microbiology2，玛丽医院，香港大学，香港，中国的
    对应德茅茬嗯[email protected]
    J.医学。微生物学，2003年5月52：397-402，DOI：10.1099 / jmm.0.05071-0
    主题：诊断，分型与鉴定

    收稿日期：2002年11月9日接受：20/01/2003网上公布：01/05/2003



    高度灵敏和快速检测已发展到量化乙型肝炎病毒（HBV）DNA的基础上，荧光共振能量转移原理和实时PCR，使用的LightCycler和一对特定的荧光杂交探针。此的LightCycler实时PCR测定法（LC-PCR）检测HBV-DNA的线性范围从每反应10 ** 1至10 ** 8份（250-2.5×10 ** 9份毫升-1），用快速35分钟PCR循环时间。该测定用两个EUROHEP HBV DNA的标准（ad和ay亚型）的验证，并表现出低测定内（<6％）和批间（<16％）的变化对两种亚型以上的数量级7的订单的全部范围。该法是临床使用血清样本120的HBsAg +个人和45名健康对照谁是阴性的HBsAg和抗-HBc评估。 HBV DNA水平分别同时使用LC-PCR和Digene公司杂交捕获II HBV DNA（HCII）试验测定这些样品中。患病率的HBV DNA的HBsAg的+血清样品中分别为95％（一百二分之一百一十四）和56％（一百二十○分之六十七）通过LC-PCR和HCII（P <0.01）。所有67 HCII阳性样品测试了正面带有LC-PCR检测，而47不和谐样品显示低水平的HBV DNA的（下降到265份毫升-1），仅由更敏感的LC-PCR法检测的。 HBV DNA作为由两个实验测量水平表现出良好的相关性（r = 0.902，P <0.001）。 HBV DNA水平是显著高于大三阳+比抗HBe +样本（中位数1.5×107 VS 4.6×104拷贝ML-1; P <0.01）。可以得出结论，这种LC-PCR检测是乙型肝炎病毒DNA的快速，灵敏和准确的测量临床上有用的。

    缩写：简历，变异系数; FRET，荧光共振能量转移;乙肝病毒，B型肝炎病毒; HCII，Digene公司杂交捕获II; LC-PCR，PCR的LightCycler。

StephenW

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