Defra - Department for Environment, Food and Rural Affairs.

Science Search

Science and Research Projects

Return to Science Search homepage   Return to Project List

USE OF THE POLYMERASE CHAIN REACTION FOR THE ANALYSIS AND ENUMERATION OF CRYPTOSPORIDIUM OOCYSTS IN DRINKING WATER - WT2225

Description
The aim of this research project is to establish whether the Polymerase Chain Reaction (PCR) could be used to streamline the analysis of Cryptosporidium species in drinking water samples.

Background

PCR is already used extensively in medical and clinical laboratories, as well as in research and forensic science 1. The Drinking Water Inspectorate (DWI) has also sponsored previous projects involving PCR2. PCR is a technique that amplifies and then identifies the DNA in a sample, one of its weaknesses is that it does not give any information on whether the DNA was associated with a viable organism. Establishing the viability of Cryptosporidium is difficult and the sensible presumption would be to err on the side of caution. This project would explore whether it could potentially be used for analysis of Cryptosporidium in water samples. The current Standing Committee of Analysts procedure for Cryptosporidium analysis 3 in drinking water involves elution of a sponge membrane, centrifugation, immune-magnetic separation (IMS), fixing, staining, mounting and enumeration using a microscope. This method is time consuming, expensive, has low specificity, with a number of other contaminants ending up on the microscope slide, and has multiple opportunities for loss of oocysts.

After the Immuno-Magnetic Separation (IMS) stage, the sample consists of about 55µl. This project would look to see if this is adequate to apply PCR, to enumerate the Cryptosporidium DNA present in the sample. Routinely, water companies would expect to find no quantifiable DNA, indicating that the treatment process has been successful in removal of oocysts. This project should examine whether the method could be set up so that it uses quantifiable PCR, which would then give water companies the information on how much DNA was recovered from the sample.

Policy and Regulatory Context

The Water Supply (Water Quality) Regulations 2016 (as amended) 4 in England and the Water Supply (Water Quality) Regulations 2018 5 in Wales specify that water companies must provide wholesome water to consumers. Public water supplies should be free of any micro-organism or parasites at a concentration or value which would constitute a potential danger to human health.

The Drinking Water Inspectorate (DWI) as the drinking water quality regulator for England and Wales and the enforcing body for adherence against the water quality standards would like to understand the potential use of PCR for the analysis of Cryptosporidium, as it may have several benefits to the water industry, such as streamlining the analytical process, providing quicker results and providing more specificity to the method.

The project will provide the Inspectorate with an understanding of the potential for use of current and new PCR methods in Cryptosporidium analysis, as well any potential limitations of the methodology.



Objective
Detailed requirements

The scope of the research project will include:
1. Literature review summary of current published research into the use of PCR methods that would be suitable for the analysis and enumeration of Cryptosporidium in drinking water.
2. A review of the use of PCR within the water industry at the moment, and how the industry may adopt the technology into their routine sample analysis.
3. Develop a PCR based method based on findings of the literature review and practical investigations
4. Duplicate analysis of spiked Cryptosporidium samples, using the current blue book method for Cryptosporidium analysis, alongside the method of PCR deemed suitable.
5. Collation and review of the results of the spiked sample analysis, to determine the benefits of the use of PCR against the current blue book method.
6. Any recommendations for the industry for the use of PCR in routine analysis of Cryptosporidium, and recommendations for any future areas of research that may be required from the results of this study.
Time-Scale and Cost
From: 2020

To: 2021

Cost: £66,080
Contractor / Funded Organisations
Public Health Wales (NHS Trust)
Keywords
Water Quality              
Water Supply