Multiplex real-time assay for the detection of 7viruses causing infections of the central nervous system

Abstract number: O48

Reijans M., Ossel J., Keijdener J., Simons G.

Objective: Molecular diagnostics play an increasingly important role in the detection of infectious agents in cerebrospinal fluids. However, the growing list of targets and the relatively small sample volumes are challenges that demand an improved molecular diagnostic approach. The MeningoFinder is a MultiFinder assay allowing the simultaneous detection of 7 viruses and 1 internal control in 1 reaction. Until now, the analysis of MultiFinder assays was based on size-fractionation, identifying each MultiFinder probe due to its specific length. Here we present an alternative approach allowing realtime detection of eight MeningoFinder probes in a single tube. The realtime detection enables a faster analysis, less handling and lowers the risk of contamination.

Method: The MeningoFinder assay is a MultiFinder assay which detects herpes simplex virus 1 and 2 (HSV1–2), human parechovirus (HPeV), cytomegalovirus (CMV), Epstein–Barr Virus (EBV), enterovirus (EV) and varicella-zoster Virus (VZV) plus an internal control in a single reaction. Each MeningoFinder probe can be distinguished based upon the specific length of each probe by size-fractionation using gel or capillary electrophoresis. We developed an alternative detection method using fluorescently labelled probes which allow specific identification of 8 MultiFinder probes in a realtime PCR machine.

Results: A large number of QCMD samples (N = 44), several enterovirus types (N = 27) and characterised clinical samples (N = 66) were analyzed using the MeningoFinder. All MeningoFinder reactions were analyzed by capillary electrophoresis and by fluorescently labelled probes in a realtime PCR machine. The results of the MeningoFinder showed a very good correlation with the expected results (>95%). Furthermore, the results of both MeningoFinder analyses showed a high degree of correlation. The realtime detection of the MeningoFinder probes decreases the analysis time and post PCR handling dramatically.

Conclusions: We developed a new assay for the realtime detection of 8 MeningoFinder probes. The realtime analysis showed a very good correlation with the conventional capillary electrophoresis analysis. In addition, the realtime detection reduced contamination risk and patient results became available more quickly. The combination of MultiFinder technology combined with realtime detection shows great potential in fast and easy multiparameter screening of clinical samples for infectious pathogens.