The role of polymerase chain reaction and its newer developments in feline medicine

Abstract

We give a brief overview on the principles of the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative competitive PCR and real-time PCR (TaqMan technology). The literature dealing with PCR and its role in the diagnosis, pathogenesis and research of infectious diseases of the domestic cat is reviewed. Cross-contaminations which occasionally occur during handling of amplified DNA may be an important problem in the PCR laboratory. In many infectious diseases, PCR results are difficult to interpret as their predictive positive and negative values are not always known. Newer assays, such as TaqMan procedures, are becoming increasingly reliable and cost-effective. It can be expected that additional knowledge on how to interpret PCR results will soon be available.

Fig 1.

Principle of PCR. A double DNA strand is denatured by heat (95°C) resulting in two single strands. The temperature is then-lowered, for example to 60°C, which allows the primers to bind to their complementary sequence. The temperature is then increased to 72°C, the optimal temperature for Taq polymerase which extends the primers at the 3′ end. After extension, four strands (two double strands) will have resulted from the two original strands. In cycle 2, the same procedure is repeated, resulting in eight strands and so on.

Fig 2.

Electrophoresis of amplified products on an agarose gel. Amplified products are loaded onto an agarose gel, separated in an electric field and, after electrophoresis, stained with ethidium bromide. Stained bands are visualised under ultraviolet illumination. DNA fragments migrate according to their size. The size of DNA bands can be determined by comparison with bands of known length. In our example M stands for ‘marker’ with bands of 1353, 1087, 872 and 603 base pairs. The markers are digestion products of the bacteriophage φX174. Lanes 1–6 show PCR products conducted with plasmids containing a fragment of the feline CCR-5 receptor (lanes 1–5); lane 6 shows no product as the plasmid did not contain the sequence of interest.

Fig 3.

Competitive quantitative FIV provirus PCR. Constant amounts of host DNA are co-amplified with varying amounts of a competitor. The competitor contains the same primer binding sites as the wild-type DNA but is shorter so that it can be separated clearly on the gel. The number of copies present in the wild-type DNA can be determined by calculation of the ‘equivalence point’, which is defined as the concentration at which the amount of competitor equals that of the wild-type DNA. At the equivalence point, the bands of the competitor and of the wild-type DNA are of identical density. In our example, the FIV proviral load of three cats was determined. DNA of each cat was co-amplified with (from left to right) 10 000, 5000, 1000, 500, 100, 50 and 0 copies of the competitor. The lanes to the right of the panel of each cat contain marker molecules. In the far right lane, 100 molecules of competitor DNA were amplified and loaded onto the gel. From visual inspection, the equivalence points for the three cats can be estimated to be between 500 and 1000 copies (first cat from left), between 50 and 100 copies (middle cat) and between 100 and 500 copies (right cat). For details, see text and (Pistello et al 1994, Allenspach et al 1996).

Fig 4.

Principle of the TaqMan PCR. In a first step (I) the probe labelled with a fluorescing marker, R, and a quencher, Q, binds to a stretch between the two primers. As long as R is covalently bound to Q, illumination by UV does not result in fluorescence. During extension (II), the 5′-3′ exonuclease activity results in hydrolysis of the probe which leads to liberation of R (III). The amount of R released during PCR can be measured directly during the entire amplification process. The amount of fluorescence that accumulates in a tube is proportional to the amount of amplified DNA. Our example shows the FCoV TaqMan principle. FCoV1229r—first primer, FCoV1128f—second primer, FCoV1200p—probe, Tfl—heat stable Thermus flavus polymerase. For details see text and Gut et al 1999.