Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania , Trypanosoma , and Plasmodium

• Published in: Diagnostic microbiology and infectious disease 2011, Vol.69 (1), p.66-73
• Main Authors: Basiye, Frank L, Schoone, Gerard J, Beld, Marcel, Minnaar, Rene, Ngeranwa, Joseph N, Wasunna, Monique K, Schallig, Henk D.F.H
• Format: Article
• Language: English
• Subjects: Angero NA™ from Mallinckrodt Baker USA; Biological and medical sciences; Buffers; Conventional PCR; DNA; DNA, Protozoan - chemistry; DNA, Protozoan - genetics; DNA, Protozoan - isolation & purification; Fundamental and applied biological sciences. Psychology; Humans; Infectious Disease; Infectious diseases; Internal Medicine; L3™ buffer; Leishmania; Leishmania - genetics; Leishmania - isolation & purification; Medical sciences; Microbiology; Nucleic Acid Amplification Techniques - methods; Parasitology - methods; Plasmodium; Plasmodium - genetics; Plasmodium - isolation & purification; Preservation, Biological - methods; RNA; RNA, Protozoan - chemistry; RNA, Protozoan - genetics; RNA, Protozoan - isolation & purification; RT-NASBA; RT-PCR; Specimen Handling - methods; Time Factors; Trypanosoma; Trypanosoma - genetics; Trypanosoma - isolation & purification
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2010.08.018

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.