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.