The application of a viability real-time PCR assay to detect viable Salmonella spp. in diarrheal stools: A spiked-stool study
Bibliographic record
Abstract
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 10 8 CFU/mL HK-cells). Spiked pooled liquid stool consisted of less stool matter and HK-cell DNA removal (Ct values ∼22, 28, 32 for 10 9 , 10 8 , and 10 7 CFU/mL) and live cell DNA detection (Ct values ∼16, 22, 30 for 10 8 , 10 6 , and 10 4 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 10 8 CFU/mL) and detected less live cell DNA (Ct values ∼26 vs 21 for 5 % vs 20 % stools with 10 6 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. • Viability real-time PCR can assess bacterial pathogen viability in clinical stools. • Stool matter and consistency can interfere with the PCR and vPCR assay performance. • Low spiked-stool concentrations (5 %) minimized false positives and false negatives.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".