Email updates

Keep up to date with the latest news and content from Malaria Journal and BioMed Central.

Open Access Highly Accessed Research

Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots

Joyce Hwang1, Juthamas Jaroensuk1, Mara L Leimanis1, Bruce Russell2, Rose McGready134, Nicholas Day34, George Snounou56*, Francois Nosten134 and Mallika Imwong37

Author Affiliations

1 Shoklo Malaria Research Unit, Mae Sot, Tak Province, Thailand

2 Singapore Immunology Network, Biopolis, Agency for Science Technology and Research (A*STAR), Singapore, Singapore

3 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

4 Centre for Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK

5 INSERM UMR S 945, Paris F-75013, France

6 Université Paris 6, Pierre & Marie Curie, Faculté de Médecine Pitié-Salpêtrière, Paris, France

7 Center for Emerging and Neglected Infectious Diseases, Mahidol University, Bangkok, Thailand

For all author emails, please log on.

Malaria Journal 2012, 11:339  doi:10.1186/1475-2875-11-339

Published: 8 October 2012

Abstract

Background

Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known.

Methods

DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number.

Results

When a 1.6 kb fragment of the parasites’ small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted.

Conclusions

The average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.

Keywords:
Archival blood spots; Plasmodium falciparum; Polymerase chain reaction