Development and Validation of the MAST ISOPLEX® VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX^®<i>VTEC</i>, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (<i>stx1/vt1</i> and <i>stx2/vt2</i>) genes in verotoxigenic <i>Escherichia coli (E. coli)</i> (<i>VTEC</i>) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX^®<i>Probes,</i> integrated into the primer set of each target. MAST ISOPLEX^®<i>Probes</i> used in the MAST ISOPLEX^®<i>VTEC</i> kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX^®<i>VTEC</i> kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.