Development and Validation of the MAST ISOPLEX[sup.®] VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 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 dev...

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Bibliographic Details
Published in:International journal of molecular sciences 2024-09, Vol.25 (18)
Main Authors: Suwara, Monika Iwona, Bennett, Matthew, Voto, Ilaria Anna Pia, Brownlie, Christopher Allan, Gillies, Elizabeth Ann
Format: Article
Language:English
Subjects:
Bacterial toxins
Biosensors
Gene amplification
Genetic aspects
Identification and classification
Methods
Online Access:Get full text
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Summary: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[sup.®] VTEC , for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2 ) genes in verotoxigenic Escherichia coli (E. coli) (VTEC ) isolates with inhibition control (IC) synthetic DNA using a single fluorophore–oligonucleotide probe, MAST ISOPLEX[sup.®] Probes, integrated into the primer set of each target. MAST ISOPLEX[sup.®] Probes used in the MAST ISOPLEX[sup.®] VTEC 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[sup.®] VTEC 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.
ISSN:1422-0067
DOI:10.3390/ijms251810067