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
:1. Introduction
2. Results
2.1. Test of Spectral Overlap between MAST ISOPLEX® Probes in the MAST ISOPLEX® VTEC Kit on the ABI7500 Fast Real-Time PCR System (Applied Biosystems)
2.2. Test of the MAST ISOPLEX® VTEC Kit on the Portable Mic qPCR Cycler (Bio Molecular Systems)
2.3. Validation of the MAST ISOPLEX® VTEC Kit on DNA Extracts from Clinical Specimens
2.4. Analytical Specificity of the MAST ISOPLEX® VTEC Kit
2.5. Validation of the MAST ISOPLEX® VTEC Kit Using INSTAND e.V. Proficiency Samples
2.6. Analytical Sensitivity of the MAST ISOPLEX® VTEC Kit
2.7. Stability of MAST ISOPLEX® VTEC Reagents
3. Discussion
4. Materials and Methods
4.1. DNA Extraction from Bacterial Cultures
4.2. DNA Synthetic Controls
4.3. Real-Time Polymerase Chain Reaction
4.4. Loop-Mediated Isothermal Amplification (LAMP) Using the MAST ISOPLEX® VTEC Kit
4.5. Test of MAST ISOPLEX® VTEC Kit on INSTAND e.V Proficiency Samples
4.6. Determination of the Analytical Sensitivity of the MAST ISOPLEX® VTEC Kit
4.7. Acceptance/Rejection Criteria
4.8. Statistical Analysis
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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(A) | ||||||
---|---|---|---|---|---|---|
Sample Number | vt1 Multiplex PCR Result | vt1 Multiplex LAMP Result | vt2 Multiplex PCR Result | vt2 Multiplex LAMP Result | vt1 or vt2 Positive (Combined PCR Results) | vt1 or vt2 Positive (Combined LAMP Results) |
1 | 39.61 | ND | 42.23 | 49.9 | + | + |
2 | 29.59 | 30.57 | 30.07 | 48.44 | + | + |
3 | ND | ND | 24.91 | 26.71 | + | + |
4 | 30.69 | 31.51 | 30.63 | 27.31 | + | + |
5 | ND | ND | 36.53 | 44.2 | + | + |
6 | 29.59 | 30.53 | 30.28 | 46.88 | + | + |
7 | ND | ND | 16.94 | 10.12 | + | + |
8 | 27.13 | 30.64 | 27.49 | 36.65 | + | + |
9 | 34.97 | 38.91 | ND | ND | + | + |
10 | ND | ND | ND | ND | − | − |
11 | ND | ND | 37.73 | ND | + | − |
12 | ND | ND | 23.84 | 14.26 | + | + |
13 | 34.39 | 28.66 | 34.38 | 28.94 | + | + |
14 | 38.3 | ND | 40.22 | 28.73 | + | + |
15 | 36.12 | 39.8 | ND | ND | + | + |
16 | ND | ND | ND | ND | − | − |
17 | 31.74 | 28.1 | ND | ND | + | + |
18 | 28.28 | 30.43 | 24.89 | 16.92 | + | + |
19 | 23.18 | 24.16 | 23.68 | 33.21 | + | + |
20 | 36.31 | 37.62 | 33.78 | ND | + | + |
21 | ND | ND | ND | ND | − | − |
22 | 25.43 | 28.38 | ND | ND | + | + |
23 | ND | ND | 26.12 | 19.78 | + | + |
24 | ND | ND | 22.16 | 27.29 | + | + |
25 | 28.74 | 30.06 | 28.54 | 34.92 | + | + |
26 | ND | N/A | 29.2 | 18.78 | + | + |
(B) | ||||||
Number of true positive results | 22 | |||||
Number of false positive results | 0 | |||||
Number of true negative results | 3 | |||||
Number of false negative results | 1 |
Clinical Performance Indicator | Calculation | Result | Result [%] |
---|---|---|---|
Sensitivity | 22/(22 + 1) | 0.96 | 96% |
Specificity | 3/(3 + 0) | 1 | 100% |
PPV | 22/(22 + 0) | 1 | 100% |
NPV | 3/(3 + 1) | 0.75 | 75% |
Accuracy | (22 + 3)/(22 + 3 + 1 + 0) | 0.96 | 96% |
Bacteria Species | Origin | ID Number | MAST ISOPLEX® VTEC LAMP Assay Results (Ct Values) | Results Interpretation | ||
---|---|---|---|---|---|---|
vt1 | vt2 | IC DNA | ||||
Escherichia coli O157 | unknown | unknown | 7.4 | 8.45 | 15.74 | Valid. Positive for verotoxigenic E. coli |
No template control | N/A | N/A | ND | ND | 12.18 | Valid. Negative for verotoxigenic E. coli |
Escherichia coli | ATCC | 11,303 | ND | ND | 12.26 | Valid. Negative for verotoxigenic E. coli |
Edwardsiella tarda | NCTC | 11,934 | ND | ND | 12.07 | Valid. Negative for verotoxigenic E. coli |
Clostridium sporogenes | ATCC | 19,404 | ND | ND | 12.4 | Valid. Negative for verotoxigenic E. coli |
Proteus mirabilis | ATCC | 29,906 | ND | ND | 12.27 | Valid. Negative for verotoxigenic E. coli |
Vibrio parahaemolyticus | ATCC | 17,802 | ND | ND | 12.25 | Valid. Negative for verotoxigenic E. coli |
Peptostreptococcus anaerobius | ATCC | 27,337 | ND | ND | 12.39 | Valid. Negative for verotoxigenic E. coli |
Bacteroides fragilis | ATCC | 25,285 | ND | ND | 12.24 | Valid. Negative for verotoxigenic E. coli |
Citrobacter freundii | ATCC | 8090 | ND | ND | 15.59 | Valid. Negative for verotoxigenic E. coli |
Proteus hauseri | ATCC | 13,315 | ND | ND | 12.41 | Valid. Negative for verotoxigenic E. coli |
Pseudomonas aeruginosa | ATCC | 12,903 | ND | ND | 12.29 | Valid. Negative for verotoxigenic E. coli |
Staphylococcus aureus | ATCC | 9144 | ND | ND | 11.64 | Valid. Negative for verotoxigenic E. coli |
Enterobacter cloacae | NCTC | 11,936 | ND | ND | 12.22 | Valid. Negative for verotoxigenic E. coli |
Yersinia enterocolitica | ATCC | 9610 | ND | ND | 12.15 | Valid. Negative for verotoxigenic E. coli |
(A) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Sample No./Target | Sample 1D: 19153 | |||||||||||
41 n1 | 41 n2 | 41 n3 | 42 n1 | 42 n2 | 42 n3 | 43 n1 | 43 n2 | 43 n3 | 44 n1 | 44 n2 | 44 n3 | |
vt1 | 13.28 | 12.46 | 11.97 | ND | ND | ND | ND | ND | ND | ND | ND | ND |
vt2 | 24.31 | 31.94 | 23.32 | ND | ND | ND | 16.62 | 18.04 | 17.45 | ND | ND | ND |
IC | 11.67 | 11.80 | 12.01 | 11.84 | 11.56 | 11.73 | 11.81 | 11.68 | 11.69 | 11.57 | 11.76 | 11.88 |
(B) | ||||||||||||
SAMPLE ID | vt1 Target Gene | vt2 Target Gene | IC Target | Result | ||||||||
1915341 | + (3/3 replicates) | + (3/3 replicates) | + (3/3 replicates) | Valid. The sample is positive for vt1 and vt2. | ||||||||
1915342 | − (3/3 replicates) | − (3/3 replicates) | + (3/3 replicates) | Valid. The sample is negative for vt1 and vt2. | ||||||||
1915343 | − (3/3 replicates) | + (3/3 replicates) | + (3/3 replicates) | Valid. The sample is positive for vt2 only. | ||||||||
1915344 | − (3/3 replicates) | − (3/3 replicates) | + (3/3 replicates) | Valid. The sample is negative for vt1 and vt2. |
(A) | ||||
---|---|---|---|---|
Concentration of vt1 Plasmid Per Reaction | Number of Copies of vt1 Plasmid Per Reaction | Total Number of Replicates | Number of Positive Replicates Obtained with vt1 Plasmid | Number of Positive Replicates Obtained with IC (Internal Control Plasmid) |
0.5 fg | 135.45 | 9 | 9 | 9 |
0.1 fg | 27.09 | 9 | 8 | 9 |
0.005 fg | 1.3545 | 9 | 1 | 9 |
0.001 fg | 0.2709 | 9 | 2 | 9 |
0.00005 fg | 0.013545 | 9 | 0 | 9 |
(B) | ||||
Concentration of vt2 Plasmid Per Reaction | Number of Copies of vt2 Plasmid Per Reaction | Total Number of Replicates | Number of Positive Replicates Obtained With Vt2 Plasmid | Number of Positive Replicates Obtained with IC (Internal Control Plasmid) |
0.5 fg | 134.7 | 9 | 9 | 9 |
0.1 fg | 26.94 | 9 | 9 | 9 |
0.005 fg | 1.347 | 9 | 2 | 9 |
0.001 fg | 0.2694 | 9 | 1 | 9 |
0.00005 fg | 0.01347 | 9 | 0 | 9 |
(C) | ||||
vt1 Plasmid | Percentile | Error | Lower | Upper |
DNA copies per reaction | 72.88 | 2.60 | 18.46 | 1987.70 |
(D) | ||||
vt2 Plasmid | Percentile | Error | Lower Limit | Upper Limit |
DNA copies per reaction | 19.07 | 2.48 | 5.71 | 1081.16 |
Controls/Samples | Target Gene (vt1- CY5 or/ and vt2- FAM) | IC TAMRA | Interpretation |
---|---|---|---|
Negative control | − | + | Valid |
Positive control | + | + | Valid |
Sample | − | + | Valid; negative for the target gene |
Sample | − | − | Invalid |
Sample | + | +/− * | Valid; positive for the target gene |
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Suwara, M.I.; Bennett, M.; Voto, I.A.P.; Brownlie, C.A.; Gillies, E.A. 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. Int. J. Mol. Sci. 2024, 25, 10067. https://doi.org/10.3390/ijms251810067
Suwara MI, Bennett M, Voto IAP, Brownlie CA, Gillies EA. 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. International Journal of Molecular Sciences. 2024; 25(18):10067. https://doi.org/10.3390/ijms251810067
Chicago/Turabian StyleSuwara, Monika Iwona, Matthew Bennett, Ilaria Anna Pia Voto, Christopher Allan Brownlie, and Elizabeth Ann Gillies. 2024. "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" International Journal of Molecular Sciences 25, no. 18: 10067. https://doi.org/10.3390/ijms251810067