Sofi Ibrahim M, Kulesh David A, Saleh Sharron S, Damon Inger K, Esposito Joseph J, Schmaljohn Alan L, Jahrling Peter B
The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA.
J Clin Microbiol. 2003 Aug;41(8):3835-9. doi: 10.1128/JCM.41.8.3835-3839.2003.
We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.
我们开发了一种高度灵敏且特异的检测方法，用于在Smart Cycler和LightCycler平台上快速检测天花病毒DNA。该检测方法基于TaqMan化学原理，以正痘病毒血凝素基因作为靶序列。使用从1975年孟加拉国天花病毒纯化的基因组DNA，在这两种仪器上估计检测限约为25个拷贝。在一项盲法研究中，用322个编码样本对该检测方法进行了评估，这些样本包括来自48种不同天花病毒分离株的基因组DNA；25种不同的骆驼痘、牛痘、鼠痘、沙鼠痘、疱疹、猴痘、黏液瘤、兔痘、浣熊痘、臭鼬痘、痘苗和水痘 - 带状疱疹病毒的毒株和分离株；以及两种立克次氏体物种DNA，其浓度大多在100 fg/微升至1 ng/微升范围内。在这322个样本中，含有从纯化病毒制剂中获得的浓度为1 fg/微升至1 ng/微升的天花病毒DNA。在Smart Cycler平台上，在检测的116个不含天花病毒的样本中检测到2个假阳性结果；即该检测方法的总体特异性为98.3%。在LightCycler平台上，检测到5个假阳性结果（总体特异性为95.7%）。在206个含有浓度范围为100 fg/微升至1 ng/微升的天花病毒DNA的样本中，在Smart Cycler平台上有8个样本被认为阴性，在LightCycler平台上有1个样本被认为阴性。因此，Smart Cycler仪器的临床敏感性为96.1%，LightCycler仪器的临床敏感性为99.5%。这些样本绝大多数来自病毒感染的细胞培养物和天花病毒感染的组织；因此，DNA材料同时包含病毒DNA和细胞DNA。在43个含有浓度范围为1 fg/微升至1 ng/微升的纯化天花病毒DNA的样本中，该检测方法在Smart Cycler和LightCycler平台上均正确检测出所有43个样本中的病毒。如果因故意或意外再次出现天花病毒感染，该检测方法可能有助于早期检测天花病毒感染。
