Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection
Feline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating x...
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MDPI AG
2025-03-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/17/3/418 |
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| author | Kotchaporn Khumtong Witsanu Rapichai Wichayet Saejung Piyamat Khamsingnok Nianrawan Meecharoen Siriluk Ratanabunyong Hieu Van Dong Supansa Tuanthap Amonpun Rattanasrisomporn Kiattawee Choowongkomon Oumaporn Rungsuriyawiboon Jatuporn Rattanasrisomporn |
| author_facet | Kotchaporn Khumtong Witsanu Rapichai Wichayet Saejung Piyamat Khamsingnok Nianrawan Meecharoen Siriluk Ratanabunyong Hieu Van Dong Supansa Tuanthap Amonpun Rattanasrisomporn Kiattawee Choowongkomon Oumaporn Rungsuriyawiboon Jatuporn Rattanasrisomporn |
| author_sort | Kotchaporn Khumtong |
| collection | DOAJ |
| description | Feline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating xylenol orange (XO) as a visual indicator for FCoV detection. The assay employed six oligonucleotide primers targeting regions of the nucleocapsid (N) gene. Under optimized conditions (65 °C, 60 min), amplification products were detected through pH-dependent colour changes in the XO dye. The RT-LAMP-XO assay exhibited high specificity for FCoV, with no cross-reactivity against other common feline viral pathogens. While the detection limit (1.7 × 10<sup>1</sup> copies/µL) was an order of magnitude higher than that of qPCR, the method offered advantages in simplicity and speed compared to existing diagnostic approaches. Although less sensitive than qPCR, the RT-LAMP-XO assay may serve as a rapid screening tool when used in combination with additional primer sets. These findings demonstrate the potential utility of XO-based RT-LAMP as a simple, visual detection method for FCoV infection. |
| format | Article |
| id | doaj-art-d1ea75f5f1dc4978917c2de760ed5faf |
| institution | OA Journals |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj-art-d1ea75f5f1dc4978917c2de760ed5faf2025-08-20T01:50:10ZengMDPI AGViruses1999-49152025-03-0117341810.3390/v17030418Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus InfectionKotchaporn Khumtong0Witsanu Rapichai1Wichayet Saejung2Piyamat Khamsingnok3Nianrawan Meecharoen4Siriluk Ratanabunyong5Hieu Van Dong6Supansa Tuanthap7Amonpun Rattanasrisomporn8Kiattawee Choowongkomon9Oumaporn Rungsuriyawiboon10Jatuporn Rattanasrisomporn11Graduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandDepartment of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandGraduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandDepartment of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandCentral Laboratory (CTL), Center for Veterinary Research and Innovation, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandDepartment of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, ThailandDepartment of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandFaculty of Veterinary Medicine, Rajamangala University of Technology Tawan-ok, Bangpra, Chonburi 20110, ThailandInterdisciplinary of Genetic Engineering and Bioinformatics, Graduate School, Kasetsart University, Bangkok 10900, ThailandDepartment of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, ThailandDepartment of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, ThailandGraduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, ThailandFeline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating xylenol orange (XO) as a visual indicator for FCoV detection. The assay employed six oligonucleotide primers targeting regions of the nucleocapsid (N) gene. Under optimized conditions (65 °C, 60 min), amplification products were detected through pH-dependent colour changes in the XO dye. The RT-LAMP-XO assay exhibited high specificity for FCoV, with no cross-reactivity against other common feline viral pathogens. While the detection limit (1.7 × 10<sup>1</sup> copies/µL) was an order of magnitude higher than that of qPCR, the method offered advantages in simplicity and speed compared to existing diagnostic approaches. Although less sensitive than qPCR, the RT-LAMP-XO assay may serve as a rapid screening tool when used in combination with additional primer sets. These findings demonstrate the potential utility of XO-based RT-LAMP as a simple, visual detection method for FCoV infection.https://www.mdpi.com/1999-4915/17/3/418feline corona virusFIPRT-LAMPXylenol orangeqPCR |
| spellingShingle | Kotchaporn Khumtong Witsanu Rapichai Wichayet Saejung Piyamat Khamsingnok Nianrawan Meecharoen Siriluk Ratanabunyong Hieu Van Dong Supansa Tuanthap Amonpun Rattanasrisomporn Kiattawee Choowongkomon Oumaporn Rungsuriyawiboon Jatuporn Rattanasrisomporn Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection Viruses feline corona virus FIP RT-LAMP Xylenol orange qPCR |
| title | Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection |
| title_full | Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection |
| title_fullStr | Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection |
| title_full_unstemmed | Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection |
| title_short | Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection |
| title_sort | colorimetric reverse transcription loop mediated isothermal amplification with xylenol orange targeting nucleocapsid gene for detection of feline coronavirus infection |
| topic | feline corona virus FIP RT-LAMP Xylenol orange qPCR |
| url | https://www.mdpi.com/1999-4915/17/3/418 |
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