Quantitative and Multiplex Detection of Pathogenic Fungi Using Padlock Probes, Generic qPCR, and Suspension Array Readout

The multiplexing qualities of padlock probes and Luminex™ technology combined with the well-established quantitative feature of qPCR were the base for a ten-plex fungal detection protocol that quantitatively reveals ten different fungal species in a single experiment. Padlock probes are oligonucleotides designed to form circular DNA when hybridizing to specific target DNA. The 5′ and 3′ regions of the probes meet and ligate only when a specific target sequence is present in the examined sample. The region of the padlock probes that separates the target-specific 5′ and 3′ ends contains general primer sequences for amplification of circularized probes by means of rolling circle amplification (RCA) and qPCR. The interspersed region also contains specific tag sequences for subsequent Luminex™ recognition.