Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Sample Collection and Sample Preparation
2.3. RNA Extraction and cDNA Synthesis
2.4. Standard Plasmid Construction
2.5. LAMP Primer Design
2.6. RT-LAMP Reaction and Optimization
2.7. Analytical Specificity of RT-LAMP-XO
2.8. Quantitative PCR (qPCR) Reaction Condition
2.9. Comparative Sensitivity of RT-LAMP-XO and qPCR Assay
2.10. Diagnosis and Statistical Analysis of FCoV Infected Clinical Sample
3. Results
3.1. Design and Verification of FCoV LAMP Primer
3.2. Optimization of RT-LAMP Conditions for FCoV Detection
3.3. Specificity of RT-LAMP-XO for FCoV Detection
3.4. Comparison of RT-LAMP-XO and qPCR on Sensitivity for FCoV Detection
3.5. RT-LAMP-XO Assay Validation
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence (5′ → 3′) | Ref. |
---|---|---|
NF3 | GCAGGYAARGGWGATGTG | This study |
NB3 | GCATCATCYTTTGGCAGG | |
NFIP (NF1c-NF2) | GTAGCRTTTGGCAGCGWTAYGG-TGCTAGAWGTRSTTCAGCYAA | |
NBIP(NB1c-NB2) | GTTCCATCAGTGTCTAGCRTG-ACTTTCACYTGRTCWCCARC | |
NLF | RGYAACGAGATCACTATCACC | |
NLB | GSYAGTCAATGGTCTGCTG | |
F9N | CGTCAACTGGGGAGATGAAC | [28] |
R9N | CATCTCAACCTGTGTGTCATC |
qPCR+ | qPCR− | Total | |
---|---|---|---|
RT-LAMP-XO + | 17 | 0 | 17 |
RT-LAMP-XO − | 0 | 60 | 60 |
Total | 17 | 60 | |
(%) Sensitivity (95% CI) | 100 (80.49% to 100%) | ||
(%) Specificity (95% CI) | 100 (94.04% to 100%) | ||
FCoV prevalence | 22.08 (13.42% to 32.98%) | ||
(%) Positive Predictive Value (95% CI) | 100 (80.49% to 100%) | ||
(%) Negative Predictive Value (95% CI) | 100 (94.04% to 100%) | ||
(%) Accuracy (95% CI) | 100 (95.32% to 100%) |
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Khumtong, K.; Rapichai, W.; Saejung, W.; Khamsingnok, P.; Meecharoen, N.; Ratanabunyong, S.; Dong, H.V.; Tuanthap, S.; Rattanasrisomporn, A.; Choowongkomon, K.; et al. Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection. Viruses 2025, 17, 418. https://doi.org/10.3390/v17030418
Khumtong K, Rapichai W, Saejung W, Khamsingnok P, Meecharoen N, Ratanabunyong S, Dong HV, Tuanthap S, Rattanasrisomporn A, Choowongkomon K, et al. Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection. Viruses. 2025; 17(3):418. https://doi.org/10.3390/v17030418
Chicago/Turabian StyleKhumtong, Kotchaporn, Witsanu Rapichai, Wichayet Saejung, Piyamat Khamsingnok, Nianrawan Meecharoen, Siriluk Ratanabunyong, Hieu Van Dong, Supansa Tuanthap, Amonpun Rattanasrisomporn, Kiattawee Choowongkomon, and et al. 2025. "Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection" Viruses 17, no. 3: 418. https://doi.org/10.3390/v17030418
APA StyleKhumtong, K., Rapichai, W., Saejung, W., Khamsingnok, P., Meecharoen, N., Ratanabunyong, S., Dong, H. V., Tuanthap, S., Rattanasrisomporn, A., Choowongkomon, K., Rungsuriyawiboon, O., & Rattanasrisomporn, J. (2025). Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection. Viruses, 17(3), 418. https://doi.org/10.3390/v17030418