Development and Application of a TaqMan‐Based One‐Step Quadruplex Reverse Transcription Real‐Time PCR (RT‐qPCR) for Differential Detection of Four Porcine Diarrhea Viruses
Bibliographic record
Abstract
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus group A (PoRVA) are recognized as major enteric viral pathogens responsible for porcine viral diarrhea. These viruses exhibit similar clinical manifestations, including vomiting, diarrhea, and dehydration, which complicate differential diagnosis. Therefore, there is an urgent need for a highly sensitive and specific diagnostic method to differentiate these pathogens. In this study, we developed a TaqMan‐based one‐step quadruplex reverse transcription real‐time PCR (RT‐qPCR) assay for the simultaneous and differential detection of these four porcine diarrhea viruses. The standard curves demonstrated correlation coefficients ( R 2 ) exceeded 0.990 across a dynamic range of 10 7.5 – 10 2.5 TCID 50 /mL, and amplification efficiency ranged from 90% to 110%. The limit of detection (LOD) were 10 1.5 TCID 50 /mL. Specificity analysis showed no cross‐reactivity with other related pathogens. The assay exhibited good repeatability, with coefficients of variation (CVs) ranging from 0.15% to 1.41% for intra‐assay and 0.09% to 2.09% for inter‐assay, respectively. Finally, this method was evaluated for its practicality in the field using 348 clinical fecal samples. The positive rates of PEDV, TGEV, PDCoV, and PoRVA were 43.68%, 0.57%, 26.44%, and 33.91%, respectively. Furthermore, the coinfection rates of PEDV/PDCoV, PEDV/PoRVA, PDCoV/PoRVA, and PEDV/PDCoV/PoRVA were14.37%, 13.51%, 2.01%, and 5.75%, respectively. Compared to singleplex RT‐qPCR assays, the quadruplex method demonstrated agreement rates ranging from 99.41% to 100% in detecting these four viral pathogens. In conclusion, the developed quadruplex RT‐qPCR assay offers a reliable, sensitive, and accurate tool for the identification of four causative agents of porcine viral diarrhea, making it suitable for clinical diagnosis, disease surveillance, and epidemiological studies.
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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".