Comparative Evaluation of an Easy Laboratory Method for the Concentration of Oocysts and Commercial DNA Isolation Kits for the Molecular Detection of Cyclospora cayetanensis in Silt Loam Soil Samples

Shipley, Alicia; Arida, Joseph; Almerı́a, S.

doi:10.3390/microorganisms10071431

Cited by 5 publications

(19 citation statements)

References 34 publications

(87 reference statements)

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“…Thus, bead-beating, emerged as the most effective method for rupturing the tough structure of the oocyst wall while also preserving genomic material by not causing excessive DNA shearing or hydrolysis. This finding aligns with other studies investigating Cryptosporidium and other parasites across various matrices ( Babaei et al, 2011; Amoah et al, 2019; Temesgen et al, 2020; Shipley et al, 2022 ). Compared to other pretreatments, bead-beating not only improved DNA extraction but also enhanced PCR sensitivity, offering a rapid, cost effective, and straightforward approach for sample preparation (Elwin et al, 2014; Lindergard et al, 2003; Scharf et al, 2020; Shipley et al, 2022).…”

Section: Resultssupporting

confidence: 92%

“…For instance, while there is no significant difference (p > 0.05) in DNA yields between the DNA kits when no pretreatment is applied, the Powersoil Pro Kit produced higher DNA yields than the QIAamp DNA Mini kit when bead-beating pretreatment was conducted. This can be explained by the higher sensitivity and inhibitor removal technology attributed to this kit, as well as its associated 10-minute homogenization step (Amoah et al, 2019; Feng et al, 2023; Shipley et al, 2022).…”

Section: Resultsmentioning

confidence: 99%

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Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater

Hachimi,

Falender,

Davis

et al. 2024

Preprint

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Cryptosporidium, a eukaryotic protozoan parasite, poses a significant public health risk as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is estimated to be largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Unlike clinical surveillance, wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized methods forCryptosporidiumspp. detection in wastewater challenges the comparability of studies. Additionally, the use of standard methods for the detection and quantification ofCryptosporidiumspp. in surface waters may not be appropriate for wastewater samples due to the more complex composition of the wastewater matrix. Thus, in this study, we evaluated methods for concentratingCryptosporidiumoocysts from wastewater, extracting DNA, and detecting its genetic markers, using wastewater seeded withC. parvumoocysts. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome particles. All methods except the Nanotrap Microbiome particles were conducted with or without additional purification via immunomagnetic separation. For DNA extractions, we tested the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit, while assessing the impact of bead beating and freeze-thaw cycles on DNA yield. Genetic detection was performed using qPCR targeting theCryptosporidium18S rRNA gene. Our results indicated that centrifugation yielded the highest oocyst recoveries (39-77%), followed by the Nanotrap Microbiome particles (24%), electronegative filtration with a phosphate buffered saline with 20% Tween 80 elution (22%), and Envirocheck HV capsule filtration (13%). IMS was found to be unsuitable due to interference from the wastewater matrix, significantly reducing recovery rates to 0.03 to 4%. DNA yields were highest with bead-beating pretreatment with either the DNeasy Powersoil Pro kit (314 gc/μL DNA) or the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA yields to under 92 gc/μL DNA, likely through DNA degradation. This study is amongst the first to compare different concentration methods, including Nanotrap Microbiome particles, and DNA extraction methods that can be utilized forCryptosporidiumspp. wastewater surveillance, highlighting the importance of method selection for accurate detection and quantification.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater

Hachimi,

Falender,

Davis

et al. 2024

Preprint

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“…Studies with the expressed goal of developing detection methods for C. cayetanensis specifically for soil are rare. Only one recent study described a method for the concentration of oocysts and molecular detection of C. cayetanensis in soil samples [ 97 ]. The study evaluated a concentration method using flotation in saturated sucrose solution and compared it to three commercial DNA isolation kits in experimentally seeded C. cayetanensis soil samples (5–10 g).…”

Section: Detection Methodsmentioning

confidence: 99%

“…Linearity of detection of the flotation method was observed and it was able to detect as few as 10 oocysts in 10 g of soil samples. This comparative study showed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is an easy, low-cost, and sensitive method that could be implemented for the detection of C. cayetanensis in environmental soil samples [ 97 ]. The flotation method was able to detect low numbers of oocysts in two different types of farm soil (10 oocysts in 10 g of either type of farm soil) without modifications.…”

Section: Detection Methodsmentioning

confidence: 99%

Cyclospora cayetanensis: A Perspective (2020–2023) with Emphasis on Epidemiology and Detection Methods

Almeria,

Chacin-Bonilla,

Maloney

et al. 2023

Microorganisms

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Cyclospora cayetanensis infections are prevalent worldwide, and the parasite has become a major public health and food safety concern. Although important efforts have been dedicated to advance toward preventing and reducing incidences of cyclosporiasis, there are still several knowledge gaps that hamper the implementation of effective measures to prevent the contamination of produce and water with Cyclospora oocysts. Some of these data gaps can be attributed to the fact that access to oocysts is a limiting factor in C. cayetanensis research. There are no animal models or in vivo or in vitro culture systems to propagate the oocysts needed to facilitate C. cayetanensis research. Thus, researchers must rely upon limited supplies of oocysts obtained from naturally infected human patients considerably restricting what can be learnt about this parasite. Despite the limited supply of C. cayetanensis oocysts, several important advances have happened in the past 3 years. Great progress has been made in the Cyclospora field in the areas of molecular characterization of strains and species, generation of genomes, and development of novel detection methods. This comprehensive perspective summarizes research published from 2020 to 2023 and evaluates what we have learnt and identifies those aspects in which further research is needed.

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“…Although there are some studies on the experimental conditions in which C. cayetanensis oocysts survive in the laboratory [ 6 , 7 ], unfortunately, there is no information on what triggers oocyst sporulation in field conditions. As is the case with other protozoa, C. cayetanensis oocysts are thought to be environmentally resistant; low numbers of oocysts with a heterogenous distribution are expected to be present in soil samples [ 8 , 9 ]. Thus, the detection of C. cayetanensis in soil usually requires concentration techniques which enrich the oocyst form of the parasite to enhance detection [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular Detection of Cyclospora cayetanensis in Two Main Types of Farm Soil Using Real-Time PCR Assays and Method Modification for Commercial Potting Mix

2023

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Cyclospora cayetanensis is a foodborne protozoan parasite that causes outbreaks of diarrheal illness (cyclosporiasis) with clear seasonality worldwide. In the environment, C. cayetanensis oocysts are very robust, and contact with contaminated soil may serve as an important vehicle in the transmission of this organism, and it is considered a risk factor for this infection. The present study evaluated a flotation concentration method, previously shown to provide the best detection results when compared with DNA isolation directly from soil samples, in two main types of farm soil, silt loam soil and sandy clay loam, as well as in commercial potting mix samples inoculated with different numbers of C. cayetanensis oocysts. The flotation method was able to detect as few as 10 oocysts in 10 g of either type of farm soil without modifications, but needed an extra wash and samples of reduced size for the processing of the commercial potting mix to be able to detect 20 oocysts/5 g. A recently modified real-time PCR method for the detection of C. cayetanensis based on a mitochondrial gene target was also evaluated using selected samples of each type of soil. This comparative study confirmed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is a sensitive method that can detect low numbers of oocysts in different types of soil.

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Comparative Evaluation of an Easy Laboratory Method for the Concentration of Oocysts and Commercial DNA Isolation Kits for the Molecular Detection of Cyclospora cayetanensis in Silt Loam Soil Samples

Cited by 5 publications

References 34 publications

Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater

Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater

Cyclospora cayetanensis: A Perspective (2020–2023) with Emphasis on Epidemiology and Detection Methods

Molecular Detection of Cyclospora cayetanensis in Two Main Types of Farm Soil Using Real-Time PCR Assays and Method Modification for Commercial Potting Mix

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