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 (␤-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.

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

“…It is worthwhile to point out that, because of the differential expression level and thus the change of hsp70 mRNA copies between different batches of C. parvum oocysts, the standard curve for hsp70 mRNA in this study is applicable to this specific fresh inoculum tested. No non-specific products were observed on the agarose gel electrophoresis following the qPCR analysis, which confirmed that the primers/probe set was specific to C. parvum hsp70 gene as also reported by others (Garcé s-Sanchez et al, 2009;Liang and Keeley, 2011).…”

“…Forward primers CPhsp70f (5 0 -AACTTTAGCTCCAGTT GAGAAAGTACTC-3 0 ), reverse primer CPhsp70r (5 0 -CATGGCT CTTTACCGTTAAAGAATTCC-3 0 ) and probe CPhsp70P (5 0 -6FAM-AATACGTGTAGAACCACCAACCAATACAACATC-BHQ1-3 0 ) targeting hsp70 gene were used (Garcé s-Sanchez et al, 2009;Liang and Keeley, 2011). Each 20-ml qPCR mixture contained 1 ml genomic DNA sample, 600 nM of each primer, 300 nM TaqMan probe, DEPC-treated DNase-free water and 1Â TaqMan universal PCR master mix [including optimized concentration of AmpliTaq gold DNA polymerase, dinucleoside triphosphates (dNTPs) with dUTP, passive reference ROX dye (Applied Biosystems; Foster City, CA)].…”

“…Three of the 9 tubes were subjected to PMA assay as described in Section 2.4, and the other three tubes were subjected to DNA extraction and qPCR procedure directly without the PMA treatment. The last three tubes were subjected to mRNA extraction using the procedure previously described by Liang and Keeley (2011) with mRNA quantified as outlined in the Section 2.5. Viable oocysts (5 Â 10 4 ) and heat killed oocysts (5 Â 10 5 ) spiked into PBS solution were used as controls.…”

“…In our previous study, we developed a rapid and highly specific RT-qPCR approach to quantify hsp70 mRNA from viable oocysts that were previously heat induced for hsp70 mRNA production in soil (Liang and Keeley, 2011). The efficient recovery of mRNA from soil, however, is more challenging than water due to the abundance of humic substances which could be coextracted with mRNA and the attachment of mRNA to naturally present components such as clay.…”

Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges

“…[3][4][5][6][7] Livestock operations and irrigation with polluted water can lead to the contamination of soil and vegetables with zoonotic faecal pathogens, in which Giardia duodenalis cysts and Cryptosporidium parvum oocysts are included. [8][9][10][11] Cryptosporidium and Giardia (oo)cysts are environmental resistant stages, and can survive at different temperatures and conditions. [12] Aerobic wastewater treatment plants use sludge activated sludge process to reduce water pollution and the presence of microorganisms.…”