Abstract
A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.
In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.
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References
Deshpande A, Gans J, Graves SW, Green L, Taylor L, Kim HB, Kunde YA, Leonard PM, Li P-E, Mark J, Song J, Vuyisich M, White PS (2010) A rapid multiplex assay for nucleic acid-based diagnostics. J Microbiol Methods 80(2):155–163. doi:10.1016/j.mimet.2009.12.001
Stucki D, Malla B, Hostettler S, Huna T, Feldmann J, Yeboah-Manu D, Borrell S, Fenner L, Comas I, Coscollà M, Gagneux S (2012) Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages. PLoS One 7(7):e41253. doi:10.1371/journal.pone.0041253
Thierry S, Hamidjaja RA, Girault G, Löfström C, Ruuls R, Sylviane D (2013) A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis. J Microbiol Methods 95(3):357–365. doi:10.1016/j.mimet.2013.10.004
Wuyts V, Mattheus W, Roosens NHC, Marchal K, Bertrand S, De Keersmaecker SCJ (2015) A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium. Appl Microbiol Biotechnol 99(19):8137–8149. doi:10.1007/s00253-015-6831-7
Wuyts V, Roosens NHC, Bertrand S, Marchal K, De Keersmaecker SCJ (2015) Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array. BioMed Res Int 2015:790170. doi:10.1155/2015/790170
European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC) (2015) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA J 13(1):3991. doi:10.2903/j.efsa.2015.3991
Scallan E, Hoekstra RM, Mahon BE, Jones TF, Griffin PM (2015) An assessment of the human health impact of seven leading foodborne pathogens in the United States using disability adjusted life years. Epidemiol Infect 143(13):2795–2804. doi:10.1017/S0950268814003185
Van den Bulcke MHG, Lievens APNR, Leunda A, MbongoloMbella EG, Barbau-Piednoir E, Sneyers MJS (2008) Transgenic plant event detection. Brussels, Belgium Patent WO 2008/092866, 2008
Van den Bulcke M, Lievens A, Barbau-Piednoir E, MbongoloMbella G, Roosens N, Sneyers M, Leunda Casi A (2010) A theoretical introduction to “combinatory SYBRGreen qPCR screening”, a matrix-based approach for the detection of materials derived from genetically modified plants. Anal Bioanal Chem 396(6):2113–2123. doi:10.1007/s00216-009-3286-7
Gödel K (1931) Über formal unentscheidbare Sätze der principia Mathematica und verwandter Systeme I. Mon Math Phys 38(1):173–198. doi:10.1007/BF01700692
Core Team R (2015) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria
Chang W, Cheng J, Allaire JJ, Xie Y, McPherson J (2015) Shiny: web application framework for R. http://CRAN.R-project.org/package=shiny. Accessed 23 Feb 2015
Acknowledgment
This work was supported by grant P4044.0103 (SalMolType) from the Scientific Institute of Public Health (WIV-ISP – RP/PJ). The National Reference Centre for Salmonella and Shigella is partially supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.
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Wuyts, V., Mattheus, W., Roosens, N.H.C., Marchal, K., Bertrand, S., De Keersmaecker, S.C.J. (2017). Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). In: Bishop-Lilly, K. (eds) Diagnostic Bacteriology. Methods in Molecular Biology, vol 1616. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7037-7_3
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DOI: https://doi.org/10.1007/978-1-4939-7037-7_3
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