用于检测锦鲤疱疹病毒的分子工具的诊断灵敏度研究。
Investigation on the diagnostic sensitivity of molecular tools used for detection of koi herpesvirus.
机构信息
Friedrich-Loeffler-Institut, Institute of Infectology, Federal Research Institute for Animal Health, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
出版信息
J Virol Methods. 2010 Feb;163(2):229-33. doi: 10.1016/j.jviromet.2009.09.025. Epub 2009 Oct 9.
Previous and new PCRs for KHV detection were compared by estimation of their sensitivity in recognizing KHV DNA in plasmids, cell culture extracted KHV DNA and total DNA obtained from field tissue samples. A modified real-time PCR (Gilad et al., 2004), combined with an internal control system (IC2, Hoffmann et al., 2006) in a duplex assay, was used as a "gold standard". The lowest reliably determined virus concentration between, 5 and 10 KHV DNA genomic equivalents, was found by real-time PCR (Gilad et al., 2004), nested PCR (Bergmann et al., 2006) and one-tube semi-nested PCR. All other published and unpublished PCRs, as well as the commercial Loopamp, recognized KHV DNA at higher concentrations only. Additionally, KHV variants, newly adapted to European conditions, which could not be detected by PCR according to Bercovier et al. (2005) were found in two field samples from carp and koi from different regions of Germany. A negative influence of sample pooling was shown with field samples tested by real-time PCR.
通过估计新的和旧的聚合酶链式反应（PCR）在识别质粒、细胞培养提取的锦鲤疱疹病毒（KHV）DNA 和从现场组织样本中获得的总 DNA 中的 KHV DNA 的灵敏度，对其进行了比较。改良的实时 PCR（吉尔阿德等人，2004 年）与内部对照系统（IC2，霍夫曼等人，2006 年）在双管检测中联合使用，作为“金标准”。吉尔阿德等人（2004 年）的实时 PCR、巢式 PCR（伯格曼等人，2006 年）和单管半巢式 PCR 发现，病毒浓度在 5 到 10 个 KHV DNA 基因组当量之间时，能够可靠地检测到最低病毒浓度。所有其他已发表和未发表的 PCR 以及商业的环介导等温扩增（LAMP），只能在更高的浓度下识别 KHV DNA。此外，根据贝尔科维耶等人（2005 年）的研究，在德国不同地区的鲤鱼和锦鲤的两个现场样本中发现了适应欧洲条件的新型 KHV 变体，这些变体无法通过 PCR 检测到。实时 PCR 检测的现场样本显示出样本混合的负面影响。
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