Rapid DNA extraction for molecular epidemiological studies of malaria
Introduction
Over the past few years PCR-based genotyping of pathogens has become a central technique applied in diagnostics and in molecular epidemiological studies. PCR amplification of a marker sequence is a convenient and practical tool in epidemiological studies, where only limited amounts of specimen, such as blood, are available. The extraction of DNA from such samples is a critical step and several methods are being used. In malaria field studies involving detection of parasite DNA and genotyping of the infecting parasites, mostly finger prick blood samples are collected (Felger et al., 1994, Beck et al., 1997). DNA is routinely isolated from these samples either by phenol/chloroform extraction or by a rapid boiling method (Foley et al., 1992). Methods for DNA preparations from blood samples in studies of epidemiological scale have to fulfil the following criteria: (1) rapid preparation and large through put, (2) high reliability, (3) production of DNA of good quality for long-term storage, (4) avoidance of cross-contamination, (5) reasonable costs.
In this study we compared four different methods of DNA preparation from samples collected during malaria field studies in order to test for their reliability, sensitivity, and ease of handling. The following methods were compared: rapid boiling method; guanidine isothiocyanate (GTC) preparation with subsequent phenol/chloroform extraction; a commercially available DNA purification kit (QIAamp® Blood Kit Cat.No.29104, Qiagen, Basle, Switzerland); and a new dipstick for DNA preparation (ISOCODE™ STIX PCR Template Preparation Dipstick, Schleicher&Schuell). The ISOCODE™ dipstick also allows storage and transportation at ambient temperature and thus was expected to be a practical tool for field studies. Because of this advantage for field studies we compared DNA obtained from the ISOCODE™ STIX for PCR amplification with DNA prepared by the other three standard extraction methods in order to assess reliability and sensitivity.
DNAs prepared by these different techniques were compared in a standard PCR amplification of the merozoite surface protein (MSP2) locus of Plasmodium falciparum. msp2 has been frequently used in genotyping studies as highly polymorphic marker (Felger et al., 1994, Contamin et al., 1996). In blood samples from areas of high malaria endemicity, multiple P. falciparum infections have been frequently observed (Felger et al., 1994). As a measurement for sensitivity we used the number of msp2 genotypes detected after PCR amplification of templates prepared by the different extraction methods. Because of the lack of fresh infected whole blood, DNA was extracted from 40 pellets of packed cells which had been stored at −20°C. Red blood cells were diluted with serum-free RPMI in order to reconstitute the red blood cell concentration. Packed cells were derived from blood samples collected in Papua New Guinea (10) and Tanzania (30) during two field studies. Parasitaemia levels within the 40 samples ranged from 1 to 739 parasites/200 leucocytes. Nine samples were microscopy negative.
Section snippets
QIAamp® Blood Kit (Cat.No.29104, Qiagen, Basle, Switzerland)
The kit was used according to the supplier's instruction. Briefly, 10 μl of blood were diluted in 190 μl phosphate-buffered saline (PBS). Two hundred microlitres of Buffer AL and 25 μl of the provided Proteinase K were added and immediately vortexed for 15 s. After incubating for 10 min at 70°C, DNA was precipitated with 210 μl 96% ethanol. The mixture was applied to a QIAamp® spin column and after centrifugation, the column was washed twice with Buffer AW. DNA was eluted with 50 μl dH2O at
Acknowledgments
Lars Henning was supported by the Daniela und Jürgen Westphal-Stiftung. This work obtained financial support from the German Science Foundation (DFG) grant no. BE1075/2-1.
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