title: Comparison of real-time PCR and MassTag PCR for the multiplex detection of highly pathogenic agents.
abstract: Multiplex PCR assays are a cost- as well as labour-effective way to analyse one sample for several pathogens simultaneously. Besides the mutual competition of the individual PCR reactions included in a multiplex PCR assay, their specific read-out displays a limiting factor for the total number of PCR reactions that can be multiplexed. In this study, two PCR systems with different read-out approaches are compared, using a pentaplex PCR assay for the detection of highly pathogenic agents. A pentaplex assay was used since five represents the current limit of real-time PCR multiplexing capacity due to the low resolution of fluorescence emission peaks of the current equipment. In contrast, MassTag PCR as a quite new technique offers the possibility to detect up to 20-30 target sequences from one reaction. After extensive and separate optimisation of the PCR protocol for both platforms, a comparative probit analysis showed good sensitivities for MassTag and real-time PCR detection. Nevertheless, the detection limits of MassTag PCR have been undercut by the real-time PCR for each target. We therefore conclude that MassTag PCR is a useful diagnostic technique for the sensitive screening for pathogens by highly multiplexed PCR assays, but cannot reach the sensitivity of real-time PCR for lower multiplexed PCR assays.
authors: Centre for Biological Security, Robert Koch Institute, Berlin, Germany. DoellingerJ@rki.de
authors: Joerg
authors: J
authors: Doellinger
authors: Kati
authors: K
authors: Schroeder
authors: Natalie
authors: N
authors: Witt
authors: Carolin
authors: C
authors: Heunemann
authors: Andreas
authors: A
authors: Nitsche
mesh_terms: D001424:Bacterial Infections / Q000175:diagnosis / Q000382:microbiology*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009345:Neisseria meningitidis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D019779:Salmonella enterica / Q000235:genetics; D012680:Sensitivity and Specificity; D013212:Staphylococcus epidermidis / Q000235:genetics
chemical_list: D017931:DNA Primers