Development and Validation of the MAST ISOPLEX Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 () 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, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 ( and ) genes in verotoxigenic () isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX integrated into the primer set of each target. MAST ISOPLEX used in the MAST ISOPLEX 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 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.