This proposal details research activities that demonstrate the feasibility and use of the integrated cell culture with rRNA in situ hybridization to detect Cryptosporidium parvum. It addresses three significant drawbacks associated with current routine methods. The proposed method is: (1) specific to the human pathogenic species, C. parvum; (2) capable of enumerating infectious oocysts; and (3) does not require direct microscopic examination by a skilled microbiologist. To produce a specific detection method, unique sequences are selected from 18s rRNA using a multiple-sequence alignment against different species of Cryptosporidium. Oligonucleotides are synthesized based on these unique sequences and labeled with a nonradioactive label. The selected probes will be evaluated for their sensitivity and specificity against different strains of Cryptosporidium. To detect viable infectors' oocysts, cell culture using HTC-8 cells is integral with in situ rRNA hybridization. To increase the signal of the chemiluminescent detection system, highly sensitive X-ray film is used to visualize individual clusters of sporozoits in the cell culture. This proposed Phase I research is an integral step towards a routine test for infectious C. parvum oocysts in finished drinking water, surface water, and groundwater.The integrated cell culture-rRNA in situ hybridization technique will detect and enumerate infectious oocysts of C. parvum without microscopic examination. It streamlines the integrated cell culture-polymerase chain reaction (PCR), is more species-specific than cell culture rRNA in situ hybridization, is comparable to cell culture focus detection method, and is more practical and economical than the integrated cell culture-PCR. Several commercial applications are possible for the integrated cell culture-rRNA in situ hybridization based on Phase I research. These applications include detecting Cryptosporidium oocysts in sources of water with Method 1622, evaluating disinfection or removal efficiency for water or wastewater treatment plants, and differentiating Cryptosporidium species.Supplemental
Keywords:small business, SBIR, monitoring, wastewater treatment, engineering
