Innovative Colorimetric Neutral Red-Based Loop-Mediated Isothermal Amplification (NR-LAMP) Assay: Transforming Rapid...

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Retroviral Research

Background:

• Feline leukemia virus (FeLV) is a global retrovirus causing severe feline diseases like leukemia and lymphoma.
• Current antigen tests for FeLV can produce false negatives, necessitating more accurate diagnostic methods.
• FeLV infection leads to immunosuppression, increasing susceptibility to other infections in cats.

Purpose of the Study:

• To develop and validate a novel, highly sensitive, and specific diagnostic assay for FeLV detection.
• To establish a cost-effective and efficient molecular method as an alternative to existing FeLV tests.
• To design a colorimetric assay for easy visual interpretation of FeLV diagnostic results.

Main Methods:

• Development of a colorimetric loop-mediated isothermal amplification (LAMP) assay using neutral red (NR-LAMP).
• Custom design of a novel LAMP primer set targeting the FeLV pol gene for enhanced specificity.
• Optimization of isothermal amplification conditions (61 °C for 40 min) and validation against polymerase chain reaction (PCR).

Main Results:

• The NR-LAMP assay achieved a detection limit of 10^0 copies/µL, demonstrating high sensitivity.
• Specificity was confirmed with no cross-amplification with other feline viruses.
• Clinical testing on 98 samples showed perfect agreement with PCR, yielding 97.3% sensitivity and 100% specificity.

Conclusions:

• The developed NR-LAMP assay is a robust, sensitive, and specific molecular diagnostic tool for FeLV.
• This assay offers superior performance and cost-effectiveness compared to existing methods.
• NR-LAMP presents a convenient and reliable alternative for FeLV inspection in clinical and commercial settings.