An Internal Reaction Control for Routine Detection of <i>Clavibacter michiganensis</i> subsp. <i>sepedonicus</i> Using a Real-Time TaqMan PCR-Based Assay
Bibliographic record
Abstract
An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 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".