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Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples

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Abstract

Purpose

The major problem of the PCR method for the search of protozoan cysts/oocysts in environmental samples is the presence of inhibitors. DNA extraction methods capable of removing inhibitory substances of environmental origin and recovering the DNA are decisive for the efficiency of PCR. This study aimed to compare the efficiency of different DNA extraction methods for the search by Cryptosporidium oocysts in water samples by molecular methods.

Methods

DNA extraction from water samples was performed using four different methods. Two methods use a chaotropic buffer to extract DNA and promote the selective binding of DNA to a silica membrane (GuSCN-silica and GFX Kit). The other method is based on the lysis and digestion of the samples in buffer and proteinase K, adsorption of impurities by an “InhibitEX” insertion matrix and purification of the DNA by a silica column (QIAamp Kit). The fourth method uses ionic and non-ionic detergents and proteinase K, to solubilize and separate the DNA from proteins, and a paramagnetic resin for DNA purification in the presence of high concentrations of guanidine ions (MAGNEX DNA Kit). Nested-PCR was performed, and the Cryptosporidium SSU rDNA gene amplified.

Results

The results demonstrated that MAGNEX and GFX commercial kits showed higher sensitivity, with detection of up to 100 oocysts/mL and 104 oocysts/mL respectively.

Conclusion

In conclusion, this study confirmed that for low-DNA environmental samples, extraction methods should include an efficient oocyst wall breaking step, and showed that the best Cryptosporidium DNA extraction methods are those that use paramagnetic resins.

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Acknowledgements

This work was supported by FAPEMIG—Foundation for Research Support of Minas Gerais State (APQ-00827-08).

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Authors and Affiliations

  1. Tropical Medicine and Infectology Postgraduate, UFTM, Cefores-Professional Education Center, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil

    Neliane Cristina Moreira

  2. Institute of Health Sciences, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil

    Marlene Cabrine-Santos

  3. Institute of Biological and Natural Sciences, Federal University of Triângulo Mineiro, Av. Getúlio Guaritá, 130, Abadia, Uberaba, MG, 38025-440, Brazil

    Márcia Benedita de Oliveira-Silva

Authors
  1. Neliane Cristina Moreira
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  2. Marlene Cabrine-Santos
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  3. Márcia Benedita de Oliveira-Silva
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Contributions

MBO-S: conceptualization, formal analysis, funding acquisition, supervision, project administration and roles/writing—original draft. MC-S: conceptualization, resources, supervision, methodology, review and editing the paper. NCM: investigation, methodology, writing-review and editing.

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Correspondence to Márcia Benedita de Oliveira-Silva.

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Moreira, N.C., Cabrine-Santos, M. & de Oliveira-Silva, M.B. Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples. Acta Parasit. 65, 995–998 (2020). https://doi.org/10.1007/s11686-020-00235-w

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  • DOI: https://doi.org/10.1007/s11686-020-00235-w

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