Fluorescence and colorimetric LAMP-based real-time detection of human pathogenic Cryptosporidium spp. from environmental samples

Abstract

Public health concerns related to pathogenic protozoa are widespread, causing significant morbidity and mortality worldwide. Due to the lack of rapid and cost-effective diagnostic methods, timely treatment and control interventions are hindered. In this study, loop mediated isothermal amplification (LAMP) methods were optimised and evaluated for the rapid detection of human pathogenic Cryptosporidium species in environmental samples. Real-time fluorescence and colorimetric detection were tested simultaneously. As a reference method, the results were compared to the well-established droplet digital polymerase chain reaction (ddPCR) method. Both LAMP-based methods successfully detected the Cryptosporidium parvum (GP60 gene) and the entire Cryptosporidium genus (SAM gene) from environmental samples with 100% specificity and no cross-reactivity. Furthermore, both colorimetric and fluorescent methods demonstrated a high sensitivity, with the same limit of detection (LOD) of 1.1 copies of C. parvum per 25 µl reaction (0.02 ng/µl). For real-time fluorescence monitoring, the LAMP reaction can be completed within 28 min, and for visual detection, within 30 min. In addition, both fluorescent and colorimetric LAMP methods showed substantial agreement with the reference method (ddPCR) used. The developed protocols were able to detect C. parvum and Cryptosporidium spp. in 50–85% (n = 60) of environmental samples (treated and untreated wastewater, sludge, and surface water) compared to 58–98% (n = 60) detected by ddPCR. The results further demonstrate that LAMP is an efficient technique for detecting Cryptosporidium spp. in environmental samples due to its simplicity, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease control and public health protection.