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

Abstract

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^®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^®Probes, integrated into the primer set of each target. MAST ISOPLEX^®Probes used in the MAST ISOPLEX^®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^®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.

Keywords: DNA/LYO3; MAST ISOPLEX® VTEC; POC; loop-mediated isothermal amplification; lyophilization; multiplex.

Figure 1

(A–D) Amplification plots generated on the ABI7500 Fast Real-Time PCR System with the MAST ISOPLEX^® VTEC kit using gBlocks containing vt1 and vt2 gene sequences. Amplification plots with gBlock sequences specific to (A) vt1 (green line), (B) vt2 (blue line), (C) vt1 (green line) and vt2 (blue line), and (D) IC only (red line). The IC control is indicated by a red line in Figure 1A–D. (positive—target DNA present; negative—target DNA absent). The vt1 and vt2 gBlocks were tested at 1 pg per reaction separately or in conjunction in the same reaction vessel. The tests were conducted in triplicate and internal control DNA (IC DNA) was included in each reaction.

Figure 1

(A–D) Amplification plots generated on the ABI7500 Fast Real-Time PCR System with the MAST ISOPLEX^® VTEC kit using gBlocks containing vt1 and vt2 gene sequences. Amplification plots with gBlock sequences specific to (A) vt1 (green line), (B) vt2 (blue line), (C) vt1 (green line) and vt2 (blue line), and (D) IC only (red line). The IC control is indicated by a red line in Figure 1A–D. (positive—target DNA present; negative—target DNA absent). The vt1 and vt2 gBlocks were tested at 1 pg per reaction separately or in conjunction in the same reaction vessel. The tests were conducted in triplicate and internal control DNA (IC DNA) was included in each reaction.

Figure 2

(A–C) Amplification plots generated on the Mic qPCR Cycler with positive control DNA plasmids from the MAST ISOPLEX^® VTEC kit. (A) CY5 channel (vt1 target gene), (B) FAM channel (vt2 target gene), (C) TAMRA (IC—inhibition control target sequence); Red lines—positive control DNA plasmids (PC); black lines—no template controls (NTCs). PC and NTCs were tested in 4 replicates.

Figure 3

Comparison of mean Ct values obtained with positive control DNA plasmids (PC) from the MAST ISOPLEX^® VTEC kit using the ABI7500 Fast Real-Time PCR System and Mic qPCR Cycler. Black bars represent mean Ct values generated with the ABI7500 Fast Real-Time PCR System and white bars represent mean Ct values obtained with the Mic qPCR Cycler. Tests on the Mic qPCR Cycler were conducted in four replicates on the same batch of product and for the ABI7500 Fast Real-Time PCR System, data were collected from 3 separate experiments conducted on 3 evaluation batches of the MAST ISOPLEX^® VTEC kit. From each batch of the product, 3 individual LAMP reaction pellets were tested with positive control DNA plasmids (PC) or molecular grade water (NTC). Mean Ct values obtained with the ABI7500 Fast Real-Time PCR System and Mic qPCR Cycler were calculated for each target sequence (vt1-CY5, vt2-FAM, IC-TAMRA in PC (inhibition control in Positive Control samples) and IC-TAMRA in NTC (inhibition control in Positive control samples in No Template Control samples)). Statistical analysis was conducted using a Two-Sample t-Test with Excel software (Microsoft^®, Redmond, WA, USA). The level of statistical significance is represented by the number of stars above the graphs (** p < 0.01, *** p < 0.001).