Using DNA Microarrays to Identify Strains and Species of Cryptosporidium

摘要

Molecular biology methods have been developed to rapidly identify Cryptosporidium sp. in water supplies. PCR primers targeting the heat shock 70 protein can specifically amplify C. parvum from other Cryptosporidium species, but distinguishing between human and animal strains requires further molecular typing methods. The primary benefit of strain typing Cryptosporidium parvum isolates lies in the ability to perform risk characterization studies to inform utilities of potential watershed issues or infrastructure problems. DNA microarrays may offer a powerful tool to identify Cryptosporidium strain and species isolates that can bridge the gap between detection for regulatory purposes and detection for epidemiological investigation. Essentially a miniaturized version of a reverse dot blot, microarrays can interrogate thousands of sites within a single gene or across multiple genes of the same organism. Using image analysis software, differences down to a single base pair mismatch can be statistically achieved. In this study, the authors designed a prototype 68 probe microarray to achieve single and double nucleotide mismatch discrimination of 7 variable positions within a 190 bp region of the C. parvum hsp70 gene. PCR was used to generate biotin or fluorescently labeled probes to hybridize to the array. Initial results with two genotype I strains and two genotype II strains indicated that the array could easily distinguish between these two genotypes. Single and double nucleotide mismatch discrimination was also possible using Cy3 labeled PCR products, but achieving this was limited by the yield of PCR products. Future studies will include other Cryptosporidium isolates and refinement of the array to address other variable regions within the hsp70 gene and other diagnostic genes. These initial developments may provide utilities with additional new and simple methods for assessing sources and types of C. parvum in watersheds.