Moderne molekularbiologische Thrombophiliediagnostik: Die Genotypisierung der Faktor-V-Leiden- und der Prothrombin-G20210A-Mutation mit dem LightCycler™

Zusammenfassung

Durch die Identifizierung zweier häufiger Ursachen der hereditären Thrombophilie, der Faktor-V-Leiden-Mutation und der Prothrombinvariante G20210A, hat sich in den letzten Jahren die diagnostische Strategie der Thrombophilieabklärung entscheidend erweitert. Bedingt durch die hohe Prävalenz dieser Mutationen und dem mit ihnen verknüpften erhöhten Thromboserisiko zählt der molekulargenetische Nachweis dieser beiden Risikofaktoren derzeit zu den am häufigsten durchgeführten DNA-Tests überhaupt. Die wachsende Bedeutung dieser Analytik stellt das Labor vor die Herausforderung, sichere und schnelle aber dennoch kostengünstige Methoden einzusetzen.

Mit dem LightCycler™ von Roche Diagnostics steht nun ein Gerätesystem zur schnellen und halbmechanisierten Mutationsdetektion zur Verfügung, das auf innovativer Fluoreszenztechnologie basiert. Es kombiniert in einem Reaktionsansatz die extrem schnelle PCR-Amplifikation mit der unmittelbar nachfolgenden Genotypisierung des PCR-Produktes durch Schmelzkurvenanalyse mittels allelspezifischer Hybridisierungssonden. Aufgrund dieser Ein-Schritt-Methodik ist beim LightCycler die Gefahr der Probenverwechslung und der Kontamination praktisch eliminiert. Darüber hinaus sind die Analysezeiten mit ca. 45 Minuten für 32 Proben extrem kurz.

Die LightCycler-Analytik erweist sich als schnell, robust und zuverlässig und ist aufgrund der geringen Bindung von Arbeitszeit hervorragend für den Einsatz in einem molekulargenetischen Labor geeignet.

Summary

Following identification of the two most frequent causes of hereditary thrombophilia, the factor V Leiden mutation and the prothrombinvariant G20210A, the strategy for the diagnosis of thrombophilia has been significantly extended. Due to the high prevalence of both mutations and the risk of thrombosis they are associated with, the molecular genetic identification of these two inherited risk factors is currently one of the most frequently performed DNA tests. In view of the growing importance of this kind of analysis there is a need for the laboratory to apply reliable, fast and cost-effective methods.

The LightCycler™ is a new PCR-system from Roche Diagnostics for the rapid and semi-automated mutation detection, based on innovative fluorescence technology. It combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping within the same run. Because genotyping with the LightCycler is a single-step analysis within a closed system the risk of sample tracking errors and end-product contamination is nearly eliminated. Furthermore, as 32 samples can be genotyped within 45 minutes, the turnaround time is very brief. Mutation detection on the LightCycler has been proved to be fast, robust and reliable. Because hands-on time required for running the genotyping assays is extremely short, the LightCycler is ideally suited for routine genotyping in a molecular genetic laboratory.

Schlüsselwörter

Prothrombin - Faktor V - Genotypisierung - Hybridisierungsproben - Fluoreszenz-Resonanz-Energietransfer

Keywords

Prothrombin - factor V - PCR - genotyping - hybridization probe - fluorescence resonance energy transfer

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