title: Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium.
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.
authors.affiliation: Centre for Clinical Research, Kenya Medical Research Institute (KEMRI), Nairobi, Kenya. fbasiye@kemri.org
authors.forename: Frank L
authors.initials: FL
authors.lastname: Basiye
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Schoone
authors.forename: Marcel
authors.initials: M
authors.lastname: Beld
authors.forename: Rene
authors.initials: R
authors.lastname: Minnaar
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Ngeranwa
authors.forename: Monique K
authors.initials: MK
authors.lastname: Wasunna
authors.forename: Henk D F H
authors.initials: HD
authors.lastname: Schallig
mesh_terms: D002021:Buffers; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010274:Parasitology / Q000379:methods*; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D011309:Preservation, Biological / Q000379:methods; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002021:Buffers; D016054:DNA, Protozoan; D016053:RNA, Protozoan