Ibrahim M S, Esposito J J, Jahrling P B, Lofts R S
Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Frederick, MD 21702, USA.
Mol Cell Probes. 1997 Apr;11(2):143-7. doi: 10.1006/mcpr.1996.0093.
A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.
对一种荧光定量5'核酸酶聚合酶链反应（PCR）检测方法进行了评估，以确定其特异性检测和区分两种正痘病毒属病毒DNA的能力。在单管检测中使用了一对靶向正痘病毒血凝素基因DNA片段的共有引物，以及两个寡核苷酸探针；每个探针都用不同的荧光报告染料和相同的淬灭染料进行标记。该检测方法基于AmpliTaq DNA聚合酶的5'→3'核酸酶活性，该活性可切割荧光素标记的杂交探针。探针切割产生特定的荧光信号，其强度可通过荧光测定法定量。在评估了各种退火温度和探针浓度的影响并对报告染料的发射强度进行归一化后，基于单碱基多态性能够检测和区分猴痘病毒和痘苗病毒的DNA。5'核酸酶PCR检测方法的灵敏度与溴化乙锭染色凝胶的灵敏度相当，但该检测方法具有更高的特异性，并且几乎无需进行繁琐的PCR后处理。
