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 machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.003 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".