Gut M, Leutenegger C M, Huder J B, Pedersen N C, Lutz H
Department of Internal Veterinary Medicine, University of Zurich, Switzerland.
J Virol Methods. 1999 Jan;77(1):37-46. doi: 10.1016/s0166-0934(98)00129-3.
A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.
我们开发了一种用于绝对定量猫冠状病毒（FCoV）的单管逆转录聚合酶链反应（RT-PCR）检测方法。该检测方法基于嗜热栖热菌（Tfl）聚合酶的5'核酸酶活性以及一种荧光探针，该探针在被切割时会产生荧光。这种荧光探针，也称为TaqMan（TM）探针（美国珀金埃尔默公司，福斯特城），是一种寡核苷酸，设计用于结合两个PCR引物之间的目标cDNA，并标记有报告染料和淬灭染料。在完整的探针中，淬灭染料通过福斯特型能量转移抑制报告染料的荧光。在聚合酶延伸步骤中，Tfl核酸外切酶活性切割杂交的探针，导致报告染料产生荧光发射。阈值循环（C(T)值）表示报告荧光的增加，分别与目标cDNA或RNA的初始量直接相关。每个循环后，通过珀金埃尔默ABI Prism 7700序列检测仪实时监测荧光。扩增完成后，将样品的C(T)值回算至由稀释标准分子扩增产生的标准曲线。以下所述的单管RT-PCR检测方法能够进行精确定量，高度灵敏、快速（无需单独的逆转录步骤和扩增后步骤）、易于操作、允许高通量样品检测、具有非常好的重现性，并且可以在低污染风险下进行。引物-探针组合的设计能够检测所有已知的FCoV毒株，也可用于检测犬冠状病毒、传染性胃肠炎病毒和猪呼吸道冠状病毒。
