Detection of Cryptosporidium parvum and C. muris Oocysts in Spiked Backwash Water using Three PCR-Based Protocols
C L Gibbons1, F M Rigi, F M Awad-Ei-Kariem
1Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK.
Abstract:
Cryptosporidium parvum is an important protozoan that was shown in recent years to be responsible for a number of water-borne outbreaks of diarrhoea. In this study, ways to improve the extraction of DNA from purified C. parvum oocysts and from backwash water (a heavily contaminated by-product of sand filtration), for use in the polymerase chain reaction (PCR), were investigated. The use of a commercial DNA purification kit reduced overall assay time as did the inclusion of an excystation step. In addition, a comparison was made between the detection limits of three PCR based protocols (standard, nested and arbitrary primed (AP)-PCR) for the detection of C. parvum and C. muris. The three PCR protocols were assayed using serially diluted DNA extracted from purified C. parvum and C. muris oocysts, and from backwash water spiked with known numbers of C. parvum oocysts. Nested PCR was the most sensitive PCR-based method tested for detecting C. parvum DNA followed by AP-PCR and standard PCR. Therefore, a system based on nested PCR for the routine monitoring of backwash water may act as a first-line detection test for the presence of C. parvum oocysts in raw water treated for human consumption.
Insights
Improved DNA extraction and nested PCR offer sensitive detection of Cryptosporidium parvum (a cause of water-borne diarrhea) in water. This method aids in routine monitoring of water for human consumption.
Area of Science:
- Environmental microbiology
- Molecular biology
- Water quality analysis
Background:
- Cryptosporidium parvum is a significant protozoan pathogen causing water-borne diarrheal outbreaks.
- Effective detection methods are crucial for monitoring water safety.
Purpose of the Study:
- To optimize DNA extraction from Cryptosporidium parvum oocysts for PCR.
- To compare the sensitivity of different PCR protocols for detecting C. parvum DNA.
- To evaluate C. parvum detection in contaminated backwash water.
Main Methods:
- DNA extraction from purified oocysts and backwash water using a commercial kit.
- Inclusion of an excystation step to improve DNA yield.
- Assay of standard, nested, and arbitrary primed (AP)-PCR protocols for C. parvum and C. muris detection.
Main Results:
- A commercial DNA purification kit and excystation step reduced assay time.
- Nested PCR demonstrated the highest sensitivity for C. parvum DNA detection.
- AP-PCR and standard PCR showed lower sensitivity compared to nested PCR.
Conclusions:
- Nested PCR is the most sensitive method for detecting Cryptosporidium parvum DNA.
- Optimized DNA extraction and nested PCR can be used for routine monitoring of backwash water.
- This approach can serve as a first-line detection test for C. parvum oocysts in raw water treatment.
