A Rapid Detection Method for Mycoplasma Pneumoniae through the Combination of Recombinase Polymerase Amplification and CRISPR-Cas12a
Bibliographic record
Abstract
Background: There is a primary pathogen responsible for Mycoplasma pneumoniae pneumonia (MPP) in children called Mycoplasma pneumoniae (MP). Patients with mild symptoms usually complain of paroxysmal dry cough by stimulation and fever, while those with severe conditions may develop bronchopneumonia, pleural effusions, and lung abscesses, and even accompanied by lesions in the nervous system or urinary system. We aimed to develop a rapid detection method for MP adhesion protein P1 by merging recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats/associated proteins (CRISPR/Cas12a) gene editing. Methods: In this study, the sequence of P1 adhesion protein region (GenBank: AF290001.1) of MP was obtained from the national center of biotechnology information (NCBI) database. Five groups of primers and probes were designed using Primer Premier 5 software (Premier Canada Inc., Charlotte, NC, USA), and the best primers were selected. Furthermore, in the meantime, the corresponding real-time fluorescence quantitative polymerase chain reaction (RT-PCR) primers were designed for subsequent validation tests to determine the reagent proportion, reaction time, reaction temperature, and primer-probe concentration of the reaction system. Finally, diseased clinical samples (strong positive, medium positive, weak positive) and healthy clinical samples (1, 2, 3) were used to verify the optimized cutting system. Results: Through the experiments, we verified that the optimal reaction time for RPA at 37 °C was 30 min, and the best product was obtained by cleaving Cas12a-crRNA for 60 min, with a result fetched within 2 h. Conclusions: This method is not only fast and convenient in operation but also ensures the specificity, rapid efficiency and visualization of the test results without relying on instruments and professional personnel. It provides a solution to the subjective judgment of the culture test method and the difficulty of smear preparation.
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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 | 0.002 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| 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".