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.

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

Supplementary files

Transparent peer review

To support increased transparency, we offer authors the option to publish the peer review history alongside their article.

View this article’s peer review history

Article information

Article type
Paper
Submitted
07 Nov 2024
Accepted
11 Jan 2025
First published
16 Jan 2025
This article is Open Access
Creative Commons BY-NC license

Sens. Diagn., 2025, Advance Article

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

R. G. Mahmudunnabi, A. S. Pannu, N. Nguyen, H. M. Stratton and M. J. A. Shiddiky, Sens. Diagn., 2025, Advance Article , DOI: 10.1039/D4SD00344F

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements