Highlights
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Both freeze-thaw and bead beating DNA extraction enable protozoa detection.
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Extraction method performance varied by parasite type and sample matrix.
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Results inform protocols for water quality and shellfish safety testing.
Keywords: Cryptosporidium parvum, Giardia duodenalis, Toxoplasma gondii, Extraction, Shellfish, Freeze-thaw, bead beating, PCR, qPCR
Abstract
We compared freeze-thaw and bead beating methods for extracting DNA from parasites in oysters and seawater. Bead beating performed better for extracting Cryptosporidium, Giardia, and Toxoplasma in seawater, while freeze-thaw yielded comparable or better detection for Cryptosporidium and Giardia in oysters. This comparison highlights relative strengths and limitations of these methods.
Cryptosporidium spp., Giardia spp., and Toxoplasma gondii are globally prevalent zoonotic parasites, transmissible through ingestion of resilient oocysts (Cryptosporidium and Toxoplasma) or cysts (Giardia), hereafter referred to as (oo)cysts in contaminated food or water (Bahia-Oliveira et al., 2017, Betancourt, 2019, Boarato-David et al., 2017). To detect tough environmental stages of these parasites in food and water, different methods have been described, including freeze-thaw cycles followed by silica-membrane spin column such as Qiagen kits (Kim et al., 2023) or bead beating used by the FastPrep kits (Cazeaux et al., 2022). To date, these methods have not been compared side by side to evaluate their performance in different environmental matrices. Here, we applied both methods in parallel to compare the FastPrep bead beating and Qiagen freeze-thaw approaches for the detection of C. parvum, G. duodenalis and T. gondii in seawater and oysters.
Cryptosporidium parvum oocysts (Iowa Isolate subtype IIa) and G. duodenalis cysts (human isolate H3, Assemblage B) were purchased from the Cryptosporidium Production Laboratory at the University of Arizona (Tucson, AZ, USA) and Waterborne™ Inc. (New Orleans, LA, USA), respectively. Sporulated T. gondii oocysts (Type II strain M4) were provided by Dr. Jeroen Saeij’s laboratory at the University of California, Davis (Davis, CA, USA). Live (oo)cysts were heat inactivated (Kim et al., 2023) before being spiked into environmental matrices to reduce biohazard risk to laboratory personnel.
Extra small Pacific oysters (Crassostrea gigas) were shucked and spiked with heat-inactivated C. parvum, G. duodenalis, and T. gondii (oo)cysts in various quantities (5–10–100–1000 (oo)cysts) using six replicates per condition. Samples were stored at 4 °C overnight, and then digested using pepsin-HCl solution as previously described (Kim & Shapiro, 2021). After digestion, the pellets were pooled together, sub-aliquoted into six uniform samples, and then resuspended in 100 μL Milli-Q water for DNA extraction (Supplementary material, Figure S1).
Seawater (40 L) was collected from a shellfish growing region and concentrated into 40 mL according to EPA Method 1623.1 (USEPA, U.S. Environmental Protection Agency, 2012). The concentrated seawater was split into 1 mL sub-aliquots, and different quantities of heat-inactivated parasites (5–10–100–1000 (oo)cysts) were spiked in six replicates. The spiked samples were stored at 4 °C overnight, and then centrifuged to obtain a 100 μL final pellet for molecular analysis (Supplementary material, Figure S2).
Two DNA extraction methods were compared in this study: i) the Qiagen DNeasy Blood & Tissue Kit (Qiagen, CA, USA) with an initial freeze-thaw cycle (4 min in liquid nitrogen followed by 4 min in boiling water), and ii) the FastDNA™ SPIN Kit for Soil (hereinafter referred to as FastPrep). To test the Qiagen protocol, DNA extraction was conducted on three of the six sample pellets (from seawater and oysters) as previously described (Kim et al., 2023). For the FastPrep protocol, the remaining three pellets (from seawater and oysters) were suspended in 978 μL sodium phosphate buffer and transferred to Lysis Matrix E tubes in FastDNA™ Spin Kit for Soil (MP Biomedicals, Germany). After adding 122 μL MT buffer, samples were homogenized using the FastPrep-24™ 5 G Instrument (40 s, amplitude 6.0, twice) and extracted according to manufacturer’s instructions. Modifications were made to enhance DNA yield according to the manufacturer's troubleshooting instructions by incubating the spin filters for 5 min at 55 °C before final centrifugation. The final DNA elution volume in both methods was 50 μL.
A multiplex nested PCR assay targeting the 18S ribosomal RNA (rRNA) gene was used to amplify DNA from targeted protozoan parasites as previously described (Supplementary material, Table S1) (Kim et al., 2023). To compare the sensitivity of different primer sets for the detection of T. gondii DNA, two additional nested PCR assays targeting the ITS1 or B1 gene were applied as previously described (Supplementary material, Table S2) (Zhu et al., 2023). We also applied TaqMan qPCR assays on a limited number of spiked samples to compare the Cq (quantification cycle) values using the QuantStudio3™ Real-Time PCR system (Applied Biosystems™, CA, USA) as described by Kim et al. (2021) (Kim et al., 2021) (Supplementary material, Table S3). The Cq values measured via qPCR from oysters or seawater spiked with 1000 (oo)cysts were compared using a non-parametric Mann-Whitney U test with significance level set at p < 0.05 using the SigmaPlot software (Systat Software, Inc., CA, USA).
The comparison of Qiagen and FastPrep methods for extracting parasite DNA from oysters and seawater demonstrated variable performance depending on the parasite type, sample concentrations, and matrices. In oysters, FastPrep enabled better T. gondii detection but failed to yield detectable G. duodenalis via multiplex PCR (Fig. 1). The failure to detect G. duodenalis in samples extracted by FastPrep may have been due to the harsh extraction dynamics of bead beating, which can damage fragile G. duodenalis cysts and affect DNA integrity. Additional factors such as differential recovery or matrix-specific PCR inhibition may also have contributed to the poor G. duodenalis amplification in oyster samples. In seawater, parasite DNA was more likely to be detected using the FastPrep extraction method (Fig. 1). When comparing gene targets for PCR detection of T. gondii, amplification at the B1 gene appeared less sensitive than detection targeting the 18S rRNA or ITS1 loci (Table 1). In qPCR analysis of the oyster samples spiked with 1000 (oo)cysts, Qiagen yielded significantly lower Cq (i.e., higher DNA concentration) values for all parasites as compared with FastPrep, while the opposite was observed in seawater (Fig. 2).
Fig. 1.
Detection of targeted protozoan pathogens using a multiplex nested PCR assay applied on oyster and seawater samples that were extracted with either the Qiagen or FastPrep DNA extraction kits.
Table 1.
Detection of Toxoplasma gondii DNA in spiked oysters and seawater extracted by Qiagen or Fastprep methods and using three different PCR assays that target different loci.
| Oocyst no./sample | PCR target | Amplification/replicates tested |
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|---|---|---|---|---|---|
| Oyster |
Concentrated seawater |
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| Qiagen | FastPrep | Qiagen | FastPrep | ||
| 1000 | 18S | 3 / 3 | 3 / 3 | 3 / 3 | 3 / 3 |
| 100 | 3 / 3 | 3 / 3 | 3 / 3 | 3 / 3 | |
| 10 | 1 / 3 | 3 / 3 | 3 / 3 | 3 / 3 | |
| 5 | 1 / 3 | 3 / 3 | 2 / 3 | 3 / 3 | |
| 1000 | ITS1 | NA* | NA | NA | NA |
| 100 | 3 / 3 | 3 / 3 | 3 / 3 | 3 / 3 | |
| 10 | 2 / 3 | 3 / 3 | 2 / 3 | 3 / 3 | |
| 5 | 1 / 3 | 3 / 3 | 2 / 3 | 3 / 3 | |
| 1000 | B1 | NA | NA | NA | NA |
| 100 | 3 / 3 | 3 / 3 | 3 / 3 | 3 / 3 | |
| 10 | 0 / 3 | 1 / 3 | 0 / 3 | 2 / 3 | |
| 5 | 1 / 3 | 3 / 3 | 1 / 3 | 3 / 3 | |
* NA (not applied): Some samples were not tested using PCR targeting the ITS1 and B1 loci because all replicates showed amplification at lower concentrations.
Fig. 2.
Quantitative cycle (Cq) of Cryptosporidium parvum, Giardia duodenalis, and Toxoplasma gondii DNA detection as measured via quantitative PCR (qPCR) in (A) oysters and (B) seawater spiked with 1000 (oo)cysts. Asterisk indicates a statistically significant difference between Qiagen and Fastprep results (p < 0.05).
Overall, FastPrep may yield more efficient detection of the targeted zoonotic parasites in seawater, while Qiagen appears to be more effective for extracting DNA from G. duodenalis in oysters, particularly at higher concentrations. Notably, our laboratory personnel report that the FastPrep workflow was more labour-intensive, involving more processing steps and handling time. This systematic method comparison underscores the strengths and limitations of each method across different matrices and parasite targets.
CRediT authorship contribution statement
Veronica Rodriguez Fernandez: Writing – original draft, Investigation. Minji Kim: Writing – review & editing, Supervision, Methodology, Formal analysis, Data curation. Karen Shapiro: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Agricultural and Food Research Initiative, Award No 2017–67017–26182 from the USDA National Institute of Food and Agriculture. Veronica Rodriguez Fernandez was supported by funding from the La Sapienza University of Rome. The authors would like to thank C. Resngit, M. Tsortos and P. Sehdev for their assistance with laboratory techniques.
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.vas.2026.100592.
Contributor Information
Veronica Rodriguez Fernandez, Email: veronica.rodriguezfernandez@uniroma1.it.
Karen Shapiro, Email: kshapiro@ucdavis.edu.
Appendix. Supplementary materials
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