Detection of viable Cryptosporidium parvum in soil by reverse transcription-real-time PCR targeting hsp70 mRNA

Abstract

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 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 of Cryptosporidium with 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, and Salmonella enterica serovar 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 6 × 10(2) oocysts g(-1) of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 10(2), 1.5 × 10(3), and 1.5 × 10(4) C. parvum oocysts g(-1) soil for sandy, loamy, and clay samples, respectively.

Fig. 1.

TaqMan reverse transcription–real-time PCR detection of IPC RNA mixed before reverse transcription with extract from sand (A), loam (B), and clay (C) samples by using extraction method 1 (solid squares), method 2 (open triangles), method 3 (solid circles), method 4 (solid triangles), and method 5 (open squares). Open circles indicate amplification of IPC RNA only. The fluorescence increase is plotted versus the cycle number of RT-PCR (plot start from cycle 26).