In this study, we described a high-throughput diagnostic system that combines very specific multiplex pathogen detection with accurate quantification over a range of target concentrations.
Circularizable ligation probes (Padlock probes) [ 20] have previously been applied successfully for multiplex pathogen detection [ 21], but do not allow quantification.
Here we describe a novel two-step technique involving multiplex PCR pathogen detection by CE-SSCP analysis followed by singleplex PCR pathogen quantification by CE-SSCP.
As an example, Wang et al. used Fe3O4-Au rods as optical probes for multiplex pathogen detection [6].
This rapid multiplex pathogen detection system showed a higher pathogen detection rate in comparison with blood culture analysis.
To date, most multiplex pathogen detection has been performed using traditional hybridization microarrays [ 8, 33].
Multiplex pathogen targets are listed in Supplementary Appendix 3. Laboratory analyses were done blinded and independent of each other.
Pathogen quantification was equally robust in single target versus mixed target assays.
Multiplex amplicon quantification consists of a multiplex polymerase chain reaction amplification of fluorescently labelled target and control amplicons, followed by fragment analysis on an ABI3730 DNA Analyser (Applied Biosystems).
In this study, we evaluated the performance of a new method for relative quantification of specific DNA sequences in routine laboratory practice, called Multiplex Amplicon Quantification (MAQ) [ 5- 9].
