1a.Objectives (from AD-416):
To investigate sensitive methods for the detection of Toxoplasma in water.
1b.Approach (from AD-416):
Different methods to detect Toxoplasma DNA and viable oocysts will be investigated using water samples intentionally contaminated with different numbers of oocysts.
Cryptosporidium species and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assays that measure gene expression have been used to understand the roles of specific genes regulating developmental stages and pathogenesis of these parasites. Key to the success of these approaches is isolating high quality messenger RNA (mRNA), which is difficult to isolate from coccidian oocysts. Although several commercial kits can theoretically provide high quality mRNA to study gene expressions in oocysts, their performance has not been evaluated on oocysts. In this study, four RNA extraction kits: RiboPure-bacteria, MasterPure RNA, RNeasy micro, and TRIzol LS reagent kits were evaluated. Results revealed that all four kits easily isolated total RNA from Cryptosporidium parvum oocysts. Analysis of total RNA quality as measured by RNA integrity number (RIN) showed sufficiently high quality values ranging from 8.4 to 9.8. However, genomic DNA (gDNA) contamination was present in the RNA extracts. Additional treatment the enzyme DNase I effectively removed gDNA contaminants, but partially degraded the RNA (RIN = 5.0 – 7.7). Isolations of total RNA from T. gondii oocysts also were attempted but were unsuccessful, yielding degraded RNA extracts. Overall, the RNeasy micro kit with additional DNase I treatment was the most effective for extracting high quality total RNA from C. parvum, but not from T. gondii oocysts, for transcriptome analyses.