Innovative Colorimetric Neutral Red-Based Loop-Mediated Isothermal Amplification (NR-LAMP) Assay: Transforming Rapid and Affordable Feline Leukemia Virus Detection

Feline leukemia virus (FeLV) is a retrovirus that globally affects both domestic and wild cats, leading to the development of leukemia, lymphoma, and immunosuppression. However, it is important to note that the daily antigen test may yield false negative results. In this study, we successfully developed the first colorimetric loop-mediated isothermal amplification (LAMP) associated with neutral red (NR-LAMP) for the detection of FeLV. The NR-LAMP assay exhibited high sensitivity and efficiency compared to the routine polymerase chain reaction (PCR) reference method. To ensure specificity, a novel LAMP primer set was custom-designed based on the <i>pol</i> gene of multiple FeLV strains, which resulted in no cross-amplification with other feline viruses. Under the optimized isothermal amplification conditions at 61 °C for 40 min, the NR-LAMP assay achieved a detection limit of 10⁰ copies/µL. Using a blind clinical test involving 98 samples, the NR-LAMP assay demonstrated perfect agreement with the reference PCR method, providing a sensitivity of 97.3% and a specificity of 100%. This proposed NR-LAMP assay surpasses other related approaches in terms of sensitivity, efficiency, and cost-effectiveness. Consequently, the colorimetric NR-LAMP reaction serves as a robust and convenient diagnostic tool for the inspection of FeLV, offering an alternative molecular method for future clinical applications and commercial utilization.