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Detection of viable Cyptosporidium parvum in soil by reverse transcription real time PCR targeting hsp70 mRNA
Citation:
Liang, Z. AND A. KEELEY. Detection of viable Cyptosporidium parvum in soil by reverse transcription real time PCR targeting hsp70 mRNA. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 77(18):6476-6485, (2011).
Impact/Purpose:
Journal article for Applied and Environmental Microbiology
Description:
Extraction of high-quality mRNA from Cryptosporidium parvum is 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 for Cryptosporidium detection in soil samples. The effectiveness of five RNA extraction methods were compared (mRNA extraction with the Dynabeads® mRNA DIRECTTM 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.TM soil RNA, and Norgen Soil RNA Purification kits) for the direct detection of Cryptosporidium with oocysts-spiked sandy, loamy and clay soils 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, and Salmonella 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 that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads® with a detection limit of 1.5×102 oocysts g-1 of sandy soil. The most efficient total RNA method was PowerSoil® with detection limits of 1.5×102, 1.5×103, and 1.5×104 C. parvum oocysts g-1 soil; for sandy, loamy, and clay samples, respectively.
