Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes

Malaria Journal

Methodology

Chris J Drakeley¹  Sophie Jones¹  Rachel Hallett¹  Colin J Sutherland¹  Patrick Corran¹  Teun Bousema²  Karina Teelen³  Cornelus Hermsen³  Theo Arens³  Robert Sauerwein³  Marga van der Vegte-Bolmer³ 

[1] Department of Immunology & Infection; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK;Department of Immunology & Infection; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;

关键词: Reverse-transcription polymerase chain reaction (RT-PCR);    Quantitative nucleic acid sequence-based amplification (QT-NASBA);    Sub-microscopic;    Gametocyte;    Detection;    Elimination;    Transmission;    Ribonucleic acid (RNA);    Filter paper;   

DOI  :  10.1186/1475-2875-11-266

 received in 2012-05-16, accepted in 2012-08-02,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundAccurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored.MethodsThree gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR).ResultsSuccessful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p < 0.0001). When papers were stored at higher temperatures, a loss in sensitivity was experienced for the FTA Classic Card but not the 903 Protein Saver Card or Whatman 3MM filter paper. The sensitivity of gametocyte detection was decreased when papers were stored at high humidity.ConclusionsThis study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

【 授权许可】

Unknown   
© Jones et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.