Going to the limits of Multiplex Real-time PCR

Olfert Landt1, Ulrich Lass1, Matthias Ballhause1, Johannes Kusters2, Pranav Patel3
1Tib Molbiol Syntheselabor GmbH, Berlin, Germany; 2Medical Microbiology, University Medical Center Utrecht, The Netherlands; 3Robert-Koch-Institut, Berlin, Germany

Common opinion is that single target PCR is more sensitive than multiplex PCR, and indeed there are more putative interactions when more primers are present in the reaction. However, multiplex PCR reduces costs and handling efforts and is particular interesting for screening purposes. Although there are many multiplex assays published, we noted that they are rarely used in clinical routine diagnostics. We present examples for diagnostic use hexaplex TaqMan assays on a LightCycler 480 II system – the current limit is the number of available dye channels – for detection of bacteria, parasites or different ESBL gene targets, and evaluation data for two gastrointestinal assays as well as some data obtained with the LightCycler Nano instrument, running even more than six assays. Since the typical feedback from laboratories was the desire to exclude single assays, or to exchange them for assays with a different specification, we developed a novel concept of modular assays, which can be combined according to the respective clinical requirement. Last not least we will present first results from multiplex Recombinase-Polymerase-Amplification (RPA) assays designed for the detection of different Coronaviruses, and discuss the exciting opportunities for an ultrafast point-of-care screening for infections.

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