SR9087 - The Development of DNA Microarray Technology for Use in Testing of Key Microbial Contaminants in Water Samples

Lead researcher

Dr. Shu Chen, Lab Services Division, University of Guelph

Objectives

1. To design and prepare DNA probes from additional pathogens suitable for arraying.
   
2. To develop and optimize multiplex PCR assays for the preparation of fluorescent target DNAs suitable for hybridization.
   
3. To evaluate the performance of the PCR/chip assays using controlled target pathogens and spiked water samples.

Expected Benefits

1. Successful completion of this project will result in a practical DNA chip that can be used for simultaneous detection of the key water and food-borne pathogens in a single sample.
   
2. Will benefit industries involved in environmental and agricultural biotechnology by providing a technology that allows the academic or industrial researchers to perform large-scale genetic analysis rapidly and cost-effectively.

Results

The key contaminants in surface and ground water resources, including Cryptosporidium, Giardia, Cyclospora, Escherichia coli, Salmonella, Listeria, and Campylobacteur, are common causes of waterborne diseases. Rapid, simple and cost effective detection of these key pathogenic contaminants is crucial in ensuring water safety and a healthy environment. The purpose of this research is to develop a microchip-based test for detection of these contaminants in water samples.

This project is a continuation/extension of our previous and ongoing research on the development of a DNA microarray-based test for detecting key food-borne bacterial pathogens including Shiga toxin producing Escherichia coli (STEC), E. coli serotype O157:H7, Campylobacter, Salmonella, Salmonella typhimurium DT104, and Listeria monocytogenes. In this project, we expanded the pathogen coverage on the bacterial microarray to include key water-borne parasites: Cryptosporidium parvum, Giardia intestinalis and Cyclospora cayetanensis. The functionality of the expanded microarray and associated procedures in testing the nine pathogens in pure cultures as well as in spiked water samples was demonstrated.

The expanded microarray (8x9 array in quadruplets) consisted of 14 probes for parasite detection and 47 probes for bacterium detection. The 14 parasite probes, that were developed from a total of 39 probes, contained the DNA sequences of the oocyst wall encoding protein (cpr1) gene of C. parvum, the ß-giardin gene (giar) of G. intestinalis and the internal transcribed spacer region (its1) of C. cayetanensis.

A fluorescent multiplex PCR assay capable of simultaneously amplifying the three parasite target genes (cpr1, giar and its1) was successfully developed. Multiplex PCR conditions were optimized. The parasite multiplex PCR assay was used together with the bacterial multiplex PCR assay that was developed in a separate project in our lab to amplify all the target genes included in the system.

The multiplex PCR-microarray system was evaluated for its specificity using 30 parasitic strains, 60 target bacteria strains and 52 non-target bacterial and parasitic strains. All of the confirmed target strains were correctly detected; all of the non-target strains were not detected. The system was also evaluated for its limit of detection using both purified cultures and spiked water samples. The system was able to detect approximately 50-100 copies of template DNA in pure cultures and about 100 copies of template DNA in spiked water sediment samples. The system was further evaluated using 30 spiked water sediment samples that were concentrated from up to1000 L of water of different sources. The samples were spiked in 21 unique combinations of the target pathogens that consisted of one, two, three or four pathogens together in one sample. The multiplex PCR-microarray system produced 21 unique hybridization images which correctly matched with the 21 unique combinations.

It was concluded that the expanded multiplex PCR-microarray system developed in this project is capable of detecting the three parasitic and the six bacterial pathogens in a single assay. It should potentially be a valuable tool for comprehensive and cost-effective water/food safety testing. Further improvement in the limit of detection of the assay and more validation studies using naturally contaminated water samples are suggested before the system can be implemented in diagnostic laboratories.

Related information

• Other projects funded through the New Directions Research Program in 2001