UMMS Affiliation

Program in Molecular Medicine

Publication Date

1-18-2018

Document Type

Article

Disciplines

Fluids and Secretions | Molecular Biology | Parasitic Diseases | Parasitology | Public Health | Tropical Medicine

Abstract

BACKGROUND: Proper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time.

METHODOLOGY: Quantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against "no preservative" controls and the "gold standard" of rapidly freezing samples at -20 degrees C. The preservation methods were compared at both 4 degrees C and at simulated tropical ambient temperature (32 degrees C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA.

CONCLUSIONS: At 4 degrees C there were no significant differences in DNA amplification efficiency (as measured by Cq values) regardless of the preservation method utilized over the 60-day period. At 32 degrees C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cq value increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4 degrees C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field.

Rights and Permissions

Copyright: © 2018 Papaiakovou et al. This is an access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

DOI of Published Version

10.1371/journal.pntd.0006130

Source

PLoS Negl Trop Dis. 2018 Jan 18;12(1):e0006130. doi: 10.1371/journal.pntd.0006130. eCollection 2018 Jan. Link to article on publisher's site

Journal/Book/Conference Title

PLoS neglected tropical diseases

Related Resources

Link to Article in PubMed

PubMed ID

29346412

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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