The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study
Viability PCR (vPCR) is a development of real-time PCR where, a viability dye is used to irreversibly remove DNA from compromised or dead cells to selectively amplify live cell DNA. A vPCR assay using PMAxx™ as the viability dye was tested on Salmonella Enteritidis-spiked diarrheal stools to study t...
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Elsevier
2025-06-01
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| Series: | The Microbe |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2950194625000743 |
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| author | Surangi H. Thilakarathna Linda Chui |
| author_facet | Surangi H. Thilakarathna Linda Chui |
| author_sort | Surangi H. Thilakarathna |
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| description | Viability PCR (vPCR) is a development of real-time PCR where, a viability dye is used to irreversibly remove DNA from compromised or dead cells to selectively amplify live cell DNA. A vPCR assay using PMAxx™ as the viability dye was tested on Salmonella Enteritidis-spiked diarrheal stools to study the effects of stool concentration (5, 10 and 20 %) and PMAxx™ treatment conditions on the vPCR assay. Three replicates were used for each stool concentration, stool type and spiked cell concentration. A higher PMAxx™ concentration or an extended dark incubation time did not improve (heat-killed) HK-cell removal (Ct values for 100 μM/10 min vs 200 μM/30 min were 29.3 ± 1.6 vs 28.7 ± 0.9 for pooled liquid stool and 30.0 ± 1.3 vs 28.0 ± 1.4 for pooled semi solid stools respectively for 108 CFU/mL HK-cells). Spiked pooled liquid stool consisted of less stool matter and HK-cell DNA removal (Ct values ∼22, 28, 32 for 109, 108, and 107 CFU/mL) and live cell DNA detection (Ct values ∼16, 22, 30 for 108, 106, and 104 CFU/mL) was similar across the 3 stool concentrations. On the other hand, more stool matter in spiked pooled semi solid stool interfered with the vPCR assay and removed less HK-cell DNA (Ct values ∼27 vs 31 for 5 % vs 20 % stools with 108 CFU/mL) and detected less live cell DNA (Ct values ∼26 vs 21 for 5 % vs 20 % stools with 106 CFU/mL) at higher stool concentrations. Consequently, 5 % stool suspensions served best for spiked stool experiments overall to minimize false positive and false negative results. Although the Salmonella spp. positive stool (n = 20) concentration did not significantly affect the PCR or vPCR results overall, comparisons between stool concentrations of each stool showed better vPCR assay performance at low soft solid stool concentrations, i.e 5 %. Small sample size with focus on a single enteric pathogen and considering 2 types of stool consistencies are limitations of this study. This study explored the opportunity of using vPCR as a viability assessment tool when culture confirmation is unavailable in a clinical diagnostic world. |
| format | Article |
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| institution | DOAJ |
| issn | 2950-1946 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
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| series | The Microbe |
| spelling | doaj-art-c55ea603a26c4aaf8fd9faf2c3d568b62025-08-20T02:54:31ZengElsevierThe Microbe2950-19462025-06-01710030610.1016/j.microb.2025.100306The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool studySurangi H. Thilakarathna0Linda Chui1Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G1C9, CanadaDepartment of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G1C9, Canada; Alberta Precision Laboratories - Public Health Laboratory (ProvLab), Edmonton, AB T6G 2J2, Canada; Corresponding author at: Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G1C9, CanadaViability PCR (vPCR) is a development of real-time PCR where, a viability dye is used to irreversibly remove DNA from compromised or dead cells to selectively amplify live cell DNA. A vPCR assay using PMAxx™ as the viability dye was tested on Salmonella Enteritidis-spiked diarrheal stools to study the effects of stool concentration (5, 10 and 20 %) and PMAxx™ treatment conditions on the vPCR assay. Three replicates were used for each stool concentration, stool type and spiked cell concentration. A higher PMAxx™ concentration or an extended dark incubation time did not improve (heat-killed) HK-cell removal (Ct values for 100 μM/10 min vs 200 μM/30 min were 29.3 ± 1.6 vs 28.7 ± 0.9 for pooled liquid stool and 30.0 ± 1.3 vs 28.0 ± 1.4 for pooled semi solid stools respectively for 108 CFU/mL HK-cells). Spiked pooled liquid stool consisted of less stool matter and HK-cell DNA removal (Ct values ∼22, 28, 32 for 109, 108, and 107 CFU/mL) and live cell DNA detection (Ct values ∼16, 22, 30 for 108, 106, and 104 CFU/mL) was similar across the 3 stool concentrations. On the other hand, more stool matter in spiked pooled semi solid stool interfered with the vPCR assay and removed less HK-cell DNA (Ct values ∼27 vs 31 for 5 % vs 20 % stools with 108 CFU/mL) and detected less live cell DNA (Ct values ∼26 vs 21 for 5 % vs 20 % stools with 106 CFU/mL) at higher stool concentrations. Consequently, 5 % stool suspensions served best for spiked stool experiments overall to minimize false positive and false negative results. Although the Salmonella spp. positive stool (n = 20) concentration did not significantly affect the PCR or vPCR results overall, comparisons between stool concentrations of each stool showed better vPCR assay performance at low soft solid stool concentrations, i.e 5 %. Small sample size with focus on a single enteric pathogen and considering 2 types of stool consistencies are limitations of this study. This study explored the opportunity of using vPCR as a viability assessment tool when culture confirmation is unavailable in a clinical diagnostic world.http://www.sciencedirect.com/science/article/pii/S2950194625000743Salmonella spp.Viability real-time PCRStool matterPropidium monoazideHeat-killed cells |
| spellingShingle | Surangi H. Thilakarathna Linda Chui The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study The Microbe Salmonella spp. Viability real-time PCR Stool matter Propidium monoazide Heat-killed cells |
| title | The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study |
| title_full | The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study |
| title_fullStr | The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study |
| title_full_unstemmed | The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study |
| title_short | The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study |
| title_sort | application of a viability real time pcr assay to detect viable salmonella spp in diarrheal stools a spiked stool study |
| topic | Salmonella spp. Viability real-time PCR Stool matter Propidium monoazide Heat-killed cells |
| url | http://www.sciencedirect.com/science/article/pii/S2950194625000743 |
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