Publication details
xMAP technology - detection of pathogenic viruses
| Authors | |
|---|---|
| Year of publication | 2018 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Most of the currently used reliable methods for molecular detection of microbiological agents rely on systems based on real-time polymerase chain reaction (qPCR). This approach offers sufficiently fast, sensitive and specific way to detect potentially present agents in various samples. Nevertheless, the huge disadvantage of qPCR is limitation of its applications for multiplex formats. There is not enough fluorescent dyes to create truly robust systems for detection of larger number of targets. Based on this disadvantage we utilized the xMAP technology and its open architecture to create high-throughput multiplex systems which could overcome this serious handicap of qPCR. The first step of analysis is ligation. In the presence of target DNA, two specific DNA probes are linked. These probes contain universal primer sequences and one of them also the specific TAG sequence. Next step is monoplex end-point polymerase chain reaction (PCR) allowing the amplification of ligated probes. One of the two used PCR primers is labelled by fluorescent dye. After the amplification, the detection using magnetic beads can follow. These beads are covered with specific oligonucleotides (anti-TAG) and each set of beads has unique absorption spectrum. Amplified probes are thus bound by their specific TAG sequence to complementary anti-TAG sequence on appropriate beads. Beads with bounded probes are eventually sorted using MAGPIX® instrument (Luminex Corporation). By this system it is theoretically possible to detect up to 50 different targets in one reaction. To meet the ever-increasing needs for rapid and reliable detection of viruses in human, food or environmental samples in our laboratory, the multiplex viral panel based on described technology will be developed. Up to now, this system includes probes for detection of hepatitis A, hepatitis E and norovirus. Validation of this system and addition of other possible targets is currently underway. |