Background and aim:
Rapid and complex diagnosis of viral, bacterial and parasitic infections is highly desirable not only in clinical practice, but also in food safety or early warning systems. Multiple Oligonucleotide Ligation followed by
PCR (MOL-PCR) is easy to set up and allows to simply add detection targets to the existing panels. The MOL-PCR technique was used in the development of the panels for the detection and identification of viruses and bacteria originating from food and feed, viral pathogens of swine, bacterial pathogens in small ruminants and biothreat agents.
MOL-PCR is based on the ligation of specific oligonucleotides in the presence of a specific target DNA. In the case of RNA viruses, reverse transcription is required. The ligation product serves as a template for the amplification with universal primers, one of which is labelled, in subsequent PCR. Visualization of PCR products is mediated by binding to magnetic fluorescent beads in a suspension array and is performed on a
MAGPIX® (Luminex) instrument. Each panel contained its own internal amplification control, which served to the differentiation of truly negative and false negative samples and control of presence of inhibitors. All the panels underwent the validation procedure adopted from the guidelines for the validation of the qPCR assays developed by FDA.
The result is a total of 5 validated panels for rapid detection and identification of the desired agents. The first includes viruses that are associated with food and water contamination - noroviruses, hepatitis A and E virus, rotaviruses and adenoviruses. The second panel is able to detect food-derived bacteria - Salmonella, Yersinia, Cronobacter, or pathogenic E. coli. Next panel is designed to detect viral agents in pigs such as African swine fever or various types of influenza. Panel dedicated to the small ruminants covers M. a. subsp. paratuberculosis, C. pseudotuberculosis, M. bovis and M. tuberculosis complex members - agents responsible for the tuberculosis and pseudotuberculosis infections. The last panel is developed for the needs of security forces and detects and typifies biothreat agents -B. anthracis, Y. pestis, F. tularensis and Brucella spp.
The developed panels have been validated for complex diagnostics of clinical material, food including water, animals and other relevant matrices. The process of optimization of MOL-PCR was established and critical points, which require a special attention in the optimization were identified. Development of all the panels was targeted with the intention to fulfill the demands placed on the diagnostic tools.