S97: Degradation of Cryptosporidium in high organic soil by reverse transcription RT- PCR targeting hsp70 mRNA

As one of the leading causes of waterborne enteric disease, Cryptosporidium parvum poses significant threat to public health. Besides water, soil can also become an important environmental source of C. parvum once polluted. Detection of viable C. parvum in soil is a key issue when considering the risk of contamination of surrounding waters. The current methods for C. parvum detection are confined to water samples. In this study we developed a reverse transcription RT-PCR method to detect viable C. parvum oocysts in soil. Five RNA extraction methods were compared for the extraction of (m)RNA from C. parvum seeded sandy, loamy and clay soils. To examine the presence of inhibitors in the extracts internal positive control (IPC) RNA was synthesized and incorporated into reverse transcription.  To mitigate RNA binding on soil components during extraction the effeciency of different facilitators (bovine serum albumin, yeast RNA, Salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella typhi) were compared and based on their performance the most efficient one, Salmonella typhi was picked. With the inclusion of Salmonella during the extraction, the most efficient extraction methods for sandy soil were, PowerSoil Total RNA and Dynabeads® mRNA DIRECT™ kit; for loamy soil, PowerSoil Total RNA kits; and clay soil, PowerSoil Total RNA kit. The detection limits were 1.5×10² (6×10²), 1.5×10³, and1.5×10⁴ C. parvum oocysts g^-1 soil, respectively.