One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for abs olute feline coronavirus (FCoV) quantitation was developed. The assay is ba sed on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fl uorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, U SE), 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 i ntact probe, the quencher dye suppresses the fluorescence of the reporter d ye by Forster-type energy transfer. During the polymerase extension steps t he Tfl exonuclease activity cleaves the hybridised probe resulting in the g eneration 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. Fluores cence is monitored in real time after each cycle by a Perkin-Elmer ABI Pris m(R) 7700 Sequence Detector. After completion of amplification, the C-T val ues of the samples are calculated back to a standard curve, generated by am plification of diluted standard molecules. The one-tube RT-PCR described be low allows precise quantitation, is highly sensitive, rapid (no separate re verse 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 primer s-probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroen teritis virus and porcine respiratory coronavirus. (C) 1999 Elsevier Scienc e B.V. All rights reserved.