1. Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium.
- Author
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Basiye FL, Schoone GJ, Beld M, Minnaar R, Ngeranwa JN, Wasunna MK, and Schallig HD
- Subjects
- Buffers, DNA, Protozoan chemistry, DNA, Protozoan genetics, Humans, Leishmania genetics, Nucleic Acid Amplification Techniques methods, Plasmodium genetics, Preservation, Biological methods, RNA, Protozoan chemistry, RNA, Protozoan genetics, Time Factors, Trypanosoma genetics, DNA, Protozoan isolation & purification, Leishmania isolation & purification, Parasitology methods, Plasmodium isolation & purification, RNA, Protozoan isolation & purification, Specimen Handling methods, Trypanosoma isolation & purification
- Abstract
Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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