Publication: Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots
1
Accepted Date
2012-10-03
Issued Date
2012-10
Copyright Date
2012
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Language
eng
ISSN
1475-2875 (electronic)
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Mahidol University
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BioMed Central
Bibliographic Citation
Hwang J, Jaroensuk J, Leimanis ML, Russell B, McGready R, Day N, et al. Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots. Malar J. 2012 Oct 8;11:339.
Suggested Citation
Hwang, Joyce, Juthamas Jaroensuk, Leimanis, Mara L., Russell, Bruce, McGready, Rose, Day, Nicholas, Snounou, George, Nosten, Francois, Mallika Imwong, มัลลิกา อิ่มวงศ์ Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots. Hwang J, Jaroensuk J, Leimanis ML, Russell B, McGready R, Day N, et al. Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots. Malar J. 2012 Oct 8;11:339.. doi:10.1186/1475-2875-11-339 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/663
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Title
Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots
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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.