Avoiding commercial kit-based DNA isolation and purification steps: a rapid method for Cryptosporidium oocyst detection†

Abstract

Current routine diagnostic tests for Cryptosporidium oocysts in water are performed in centralised laboratories using the National Association of Testing Authorities (NATA) approved USEPA Method 1623.1. This method uses fluorescent microscopy, which suffers from artefacts and false positive responses from contaminating oocyst-size particles. Additionally, existing molecular detection methods based on real-time PCR (qPCR) require purified nucleic acid, primarily relying on laborious, time-consuming, and expensive centralised laboratory-based DNA isolation procedures using commercial kits. Both the microscopy and PCR-based molecular techniques are not suitable for rapid detection due to the nature of the experiment and instrumentation. This study reports a rapid and simple method that eliminates the need for multi-step DNA isolation and purification procedures. The method involves the direct heat lysis of magnetically isolated Cryptosporidium oocysts from water samples, followed by a loop-mediated isothermal amplification (LAMP)-based detection. The analytical performance of this assay reveals a LOD of 0.17 copies per μL of genomic DNA (gDNA) with a dynamic range from 1.05 × 10⁴ copies per μL to 1.05 copies per μL. We simulated the matrix effect by putting mud into tap water and spiked oocysts to demonstrate the practical applicability of the assay. The designed LAMP detected as low as 5 and 10 oocysts per 10 mL of tap water without and with simulated matrices, respectively. The ultrasensitive nature of this assay can be attributed to its acceleration due to targeting an intron-less gene. We propose that this simple and rapid method can be extended to detect various types of pathogens.