Real-Time PCR

Carpegen specializes in the development of individual real-time PCR analyses.

The company launched a test based on real-time PCR for the identification of 6 periodontal pathogens as well as the total bacterial load in July 2003 under the brand name Carpegen^® Perio Diagnostics (formerly: meridol^® Perio Diagnostics).

Real-time PCR is highly sensitive and specific and permits exact quantification of the pathogenic species.

Further testing systems based on real-time PCR will be launched in the next few years. Because of its unique ability to quantify, Carpegen uses this technology to monitor the progression of infection (e.g. under antibiotic therapy).

Technology

Real-time PCR Real-time PCR permits the reliable and precise quantification of bacteria in subgingival samples. At the same time, it is highly specific and sensitive. Isolated and purified bacterial DNA (genetic material) is the starting point.

Conventional PCR (polymerase chain reaction) is a molecular analytical method that detects tiny amounts of DNA by amplification. An enzyme (Taq polymerase) amplifies desired species-specific gene fragments (target sequences). Every target sequence uses specific primers (short DNA fragments) as a starting point for Taq polymerase, which synthesizes numerous copies of the target sequence in an exponential process. The result of the reaction can be visualized by additional laboratory methods, e.g. gel electrophoresis.

However, conventional PCR, using detection at the end, only delivers very limited information about the number of bacteria present in a sample. Reliable quantification is impossible.

Real-time PCR, on the other hand, permits accurate quantification of target sequences using a fully automated validated process. By monitoring the progress of the amplification reaction, the exact number of bacteria in a sample can be determined.

In addition to primers specific for the respective pathogen DNA, real-time PCR uses another species-specific DNA fragment (TaqMan^® probe) in the same reaction. This fragment binds within the target sequence. The additional probe makes this method highly specific. While the target sequence is amplified, the exonuclease activity of the Taq polymerase cleaves the fluorescently labeled TaqMan^® probe from the target sequence and deletes it. This degradation of the probe releases a fluorescent signal which can be monitored online in the reaction by automatic laser detection. The intensity of the fluorescent signal thus represents the amount of product formed and is directly proportional to the initial amount of the pathogen in the patient sample.

In contrast to conventional PCR methods, no further laboratory steps are required for real-time PCR.