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Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus
Published on: May 18, 2020
Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay
Wichayet Saejung1,2, Kotchaporn Khumtong1,2, Witsanu Rapichai2,3
1Graduate Program in Animal Health and Biomedical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
Insights
A new neutral red loop-mediated isothermal amplification (NR-LAMP) assay offers a rapid, sensitive, and specific method for detecting feline immunodeficiency virus (FIV) proviral DNA. This colorimetric approach provides a promising alternative for diagnosing FIV in cats.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Feline Infectious Diseases
Background:
- Feline immunodeficiency virus (FIV) causes immunodeficiency in cats worldwide.
- Current antibody detection methods for FIV have limitations, including potential false-positive results.
Purpose of the Study:
- To develop a one-pot loop-mediated isothermal amplification (LAMP) assay integrated with neutral red (NR-LAMP) for FIV proviral DNA detection.
- To establish a rapid, specific, and sensitive diagnostic tool for FIV.
Main Methods:
- A gag gene-based, one-pot NR-LAMP assay was developed for colorimetric detection of FIV proviral DNA.
- Optimization of amplification temperature and time was performed.
- Specificity and sensitivity were evaluated using clinical samples and comparison with conventional PCR.
Main Results:
- The NR-LAMP assay demonstrated optimal amplification at 65°C for 40 minutes.
- High specificity (98.44%) was confirmed, with no cross-amplification with other feline viruses.
- The assay achieved a detection limit of 4.2 × 10^1 copies/μL and 100% sensitivity compared to PCR in 80 clinical samples.
Conclusions:
- The developed NR-LAMP assay is a suitable alternative for the molecular diagnosis of FIV-infected cats.
- This method offers a convenient, rapid, and sensitive approach for FIV detection.
Background And Aim:
Feline immunodeficiency virus (FIV) is a retroviral pathogen globally responsible for immunodeficiency disease in cats. However, the current diagnosis based on antibody detection has limitations and can also produce false-positive results. This study aimed to develop a one-pot loop-mediated isothermal amplification (LAMP) process integrated with neutral red (NR-LAMP) assay for detection of FIV proviral DNA.
Materials And Methods:
We developed a one-pot, gag gene-based NR-LAMP for convenient, rapid, specific, and sensitive colorimetric inspection of FIV proviral DNA.
Results:
The developed NR-LAMP was capable of amplifying at an optimum temperature of 65°C for 40 min. No cross-amplification was detected between FIV and other feline viruses tested, indicating the high specificity (98.44%) of the novel FIV-LAMP primer. Our NR-LAMP assay has a detection limit of 4.2 × 101 copies/μL. A total of 80 clinical samples with a background of FIV infection were collected and tested using the proposed method. The NR-LAMP assay showed a high sensitivity of 100% compared to conventional polymerase chain reaction assay.
Conclusion:
These results support the suitability of NR-LAMP as a potential future alternative clinical molecular approach for further use in the diagnosis of FIV-infected cats.
