Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay

Abstract

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. Keywords: feline immunodeficiency virus, loop-mediated isothermal amplification, molecular diagnosis, neutral red.