Multiplex xMAP Technology for a Complex Detection of Pathogenic Agents Significant for the Protection of Human and Animal Health

Abstract
Early detection of pathogenic agents in the human and veterinary field is one of the main and key moments for the treatment of infectious diseases. For this we use molecular biological methods, such as a polymerase chain reaction (PCR). Methods of molecular diagnostics are generally focused on finding specific or virulence genes and therefore the subsequent design of probes which are necessary for detection of the selected pathogen. Also specific mutation in gene, or single nucleotide polymorphism (SNP) can be used in probe design. Nowadays, a large number of pathogens need to be identified during one reaction in a short time. One of the possible multiplex sample analysis is xMAP technology (x = analyte, MAP = Multi Analyte Profiling). xMAP technology is based on a combination of existing laboratory methods such as PCR, flow cytometry and ELISA, and enables detection of more than 50 different analytes (nucleic acids) simultaneously during one reaction. In this case, a multiplex oligonucleotide ligation (MOL) is performed prior to the PCR in which there is only one pair of universal primers. One of the primers is labeled with a fluorescent dye. xMAP technology uses magnetic microspheres with a special spectral address, to which the analyte is then binds specifically with the sample.
In our laboratory, we focused on obtaining a comprehensive protocol for the MOL-PCR method followed by MagPix analysis. The assay is suitable for rapid multiplex detection of bacteria, parasites and also viruses in real samples. The final result of this work is the creation of individual multiplexing systems (detection panels). At present, we have developed diagnostic panels for the multiplex detection of 5 bacteria (Campylobacter jejuni, Escherichia coli – EHEC, Yersinia enterocolitica, Listeria monocytogenes, Salmonella enterica) and 4 parasites (Toxoplasma gondii, Taenia saginata, Trichinella spiralis, Giardia intestinalis). We continue to develop a panel for identification of pathogens that can be used in bioterrorism: Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis.
This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.