Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.