Collection of blood samples on filter paper has a history of more than 40 years.1 This simple and feasible collection and storage method has been adopted for broad use in diagnostic screening,1 drug monitoring,2 and genetic analysis,3 being particularly suitable for molecular epidemiologic studies in remote areas with tropical climate, where transport and storage conditions are often not optimal. Loss of sensitivity of polymerase chain reaction (PCR)-based methods due to field conditions has previously been reported,4 a probable consequence of lower purity, stability, and integrity of the extracted DNA from filter paper samples. A crucial problem with sample collection on filter paper is the limitation in sample volume that can be incorporated into the extraction procedure. An optimal DNA isolation process is therefore a pre-requisite for quality and reproducibility of data.
The new method described here was developed when DNA was extracted from archived blood samples on filter paper from clinically and/or microscopically confirmed individuals using Chelex® (Bio-Rad Laboratories, Hercules, CA), methanol, and boiling methods. The present study was performed to validate this new TE (Tris-EDTA) buffer-based DNA isolation method against the two established methods based on methanol5,6 and Chelex®.7,8
A total of thirty blood samples stored on two different filter papers were analyzed in duplicate in February 2004. Fifteen samples collected on 3MM® Whatman (Brentford, United Kingdom) filter paper originated from a clinical malaria drug trial in a holoendemic area in Zanzibar in November 2002, and 15 samples collected on 903® Schleicher & Schuell (Dassel, Germany) filter paper originated from a cross-sectional malariometric survey in a mesoendemic area in Mali in September 2001. The studies were reviewed and approved by the two local ethical committees and the Karolinska Institute, and all participants provided informed consent. Samples were chosen to include infections with different parasite densities, established by microscopy, that ranged from 0 to 55,360/μL of blood (Table 1). Three samples of the two respective filter papers were negative by microscopy. To ensure comparable quantities of blood, punches of same dimension (4 mm in diameter) were taken from individual dried blood spots (six punches from each spot) using a sterile biopsy punch (Kai Industries Co., Ltd., Oyana Seki City, Japan). We consider that potential differences in the amount of DNA gained from punches of individual blood sample may result from differences in extraction effectiveness. The three DNA extraction methods used are described below.
Tris-EDTA buffer-based extraction.
Tris-EDTA buffer, composed of 10 mM Tris, pH 8.0 (Tris-base plus Tris-HCl) and 0.1 mM EDTA in distilled water, was prepared and kept at room temperature. Each filter paper punch was placed in an Eppendorf (Hamburg, Germany) tube, soaked in 65 μL of TE buffer, and incubated at 50°C for 15 minutes. The punches were than pressed gently at the bottom of the tube several times, using a new pipette tip for each punch and heated at 97°C for 15 minutes to elute the DNA. The liquid condensing on the lid and the wall of the tubes were removed by a short centrifugation (2–3 seconds). The DNA extract was kept at 4°C for use within a few hours or stored at −20°C.
Methanol extraction.6
Each filter paper punch was soaked in 125 μL of methanol. After incubation at room temperature for 15 minutes, the methanol was removed and the samples were dried before adding 65 μL of distilled water. The punches were mashed using a new pipette tip for each punch and heated at 97°C for 15 minutes to elute the DNA.
Chelex® extraction.8
Each filter paper punch was incubated overnight at 4°C in 1 mL of 0.5% saponin in phosphate-buffered saline (PBS). The punches were washed for 30 minutes in PBS at 4°C, transferred into new tubes containing 25 μL of stock solution (20% Chelex-100 and 75 μL of distilled water), and vortexed for 30 seconds. The tubes were heated at 99°C for 15 minutes to elute the DNA, vortexed, and centrifuged at 10,000 × g for 2 minutes. The supernatants (65 μL) were transferred into new tubes.
The three sets of DNA generated from each sample by the three respective methods were analyzed together in the same PCR including negative and positive controls. The same procedure was performed in duplicate at a different time point.
Detection of Plasmodium falciparum parasites by PCR was performed by nested amplification of merozoite surface protein 2 (msp 2), as previously described.9 This method is widely used in molecular epidemiologic studies and drug trials to determine the number and types of parasite genotypes of P. falciparum infections. The highly polymorphic, single-copy msp 2 gene has been found to be the most informative single marker frequently used to assess mean number of genotypes per infection, i.e., multiplicity of infection.10 The polymorphic block 3 of msp 2 was targeted with outer primers in a first reaction, followed by two separate amplifications with primers specific for the two allelic families of msp 2 (FC27 and IC). The final concentration of the PCR mixture for the nest 1 amplification was 1× PCR buffer, 2 mM MgCl2, 125 μM dNTP, 250 nM of each primer (F/R), and 0.02 units/μL of Taq polymerase. Three microliters of DNA template was added to a reaction volume of 18 μL. The nest 2 amplifications were carried out in a 20-μL reaction volume containing 1× PCR buffer, 1 mM MgCl2, 125 μM dNTP, 125 nM of each primer (F/R), and 0.02 units/μL of Taq polymerase. The product of the first amplification was used as the template for the second PCR (1.2 μL/reaction). An overlay of 40 μL of mineral oil was added to each reaction tube in both amplifications. The PCR products were subjected to electrophoresis on 2% Meta-Phor® gels (Cambrex Bio Science, Rockland, ME), stained with ethidium bromide, and visualized by ultraviolet transillumination.
The sensitivity of detection varied for the different extraction methods and the results are shown in Table 1. Plasmodium falciparum parasites were detected by PCR in all samples by at least one method. Analyses of samples collected on 3MM® Whatman filter paper and with parasites detected by microscopy resulted in a high sensitivity (83–100%) for all extraction methods. For microscopy-negative samples, the sensitivity was 0% for the methanol method, 67% for the Chelex® method, and 100% for the TE buffer method. The overall sensitivity for samples collected on 903® Schleicher & Schuell filter paper was lower (33–89% for the methanol method, 0% for the Chelex® method, and 83–100% for the TE buffer, where the lower values represents the microscopy-negative samples).
The total number of msp 2 alleles, the multiplicity of infection, i.e., mean number of msp 2 alleles in all samples, and reproducibility of results for samples run in duplicate are shown in Table 2. Differences in multiplicity of infection obtained after the use of the three extraction methods were evaluated within the same type of filter paper by a t-test. For the samples collected on 3MM® Whatman filter paper, the total number of msp 2 alleles were the same with TE buffer and Chelex® methods, with a statistically significant higher multiplicity of infection compared with the methanol method, i.e., 2.00 versus 1.40 (P = 0.007, by t-test for dependent samples). Analysis of the 903® Schleicher & Schuell filter paper resulted in a higher total number of msp 2 alleles using the TE buffer compared with the methanol method, with a mean multiplicity of infection of 2.07 versus 1.27 (P = 0.017, by t-test for dependent samples). The Chelex® method showed negative results in all samples on this filter paper.
All samples were extracted in duplicate by the three respective methods and the reproducibility, based on the number and size of alleles, was highest for the TE buffer extraction method for both 3MM® Whatman and 903® Schleicher & Schuell filter paper (Table 2).
Thus, the TE buffer method was superior to the methanol and Chelex® methods both in sensitivity and reproducibility when performed on the two filter paper types stored for 15 and 29 months, respectively. Differences in the sensitivity of PCR detection of P. falciparum using DNA templates extracted by different methods have previously been described.10–12 Filter paper type, duration of storage, and parasite densities are other factors potentially influencing sensitivity.4 The lower sensitivity and reproducibility from 903® Schleicher & Schuell filter paper blood spots may be explained by the 14-month longer storage of these samples. It is worth noting that Chelex® extraction of samples on this filter paper generated negative results, whereas 3MM® Whatman filter paper samples extracted under the exact same conditions generated excellent results. Some extraction methods may thus be more suitable for a particular filter paper type.
In the developed TE buffer protocol, DNA was extracted a few hours prior to the PCR amplification, which may be important for optimal results. The effect on long-term storage on the quality of DNA extracted by the TE buffer method still needs to be evaluated.
The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive and cost-effective DNA extraction methods. The TE buffer-based DNA extraction method described in this report has shown superior results, compared with two standard methods for extraction of archived blood samples on two different types of filter paper, that are independent of parasite density and duration of storage. The new method is rapid, simple, and inexpensive, and confers a reduced risk for cross-contamination due to minimum manipulation of samples during extraction. The described method may therefore represent a useful tool in molecular epidemiological studies.
Sensitivity of Plasmodium falciparum detection by polymerase chain reaction with different DNA extraction methods
| Methanol method | Chelex method | TE buffer method | |||||
|---|---|---|---|---|---|---|---|
| Filter paper | Parasite density* | Positive/tested† | Sensitivity (%) | Positive/tested† | Sensitivity (%) | Positive/tested† | Sensitivity (%) |
| * Parasites μl established by microscopy. | |||||||
| † Individual blood samples analyzed in duplicate. | |||||||
| ‡ Samples from a clinical drug trial in east Africa (November 2002). | |||||||
| § Samples from a cross sectional survey in west Africa (September 2001). | |||||||
| Whatman 3MM‡ | 0 | 0/6 | 0 | 4/6 | 67 | 6/6 | 100 |
| 310–620 | 5/6 | 83 | 6/6 | 100 | 6/6 | 100 | |
| > 1,000 | 17/18 | 94 | 18/18 | 100 | 18/18 | 100 | |
| Overall | 22/30 | 73 | 28/30 | 93 | 30/30 | 100 | |
| Schleicher & Schuell§ | 0 | 2/6 | 33 | 0/6 | 0 | 5/6 | 83 |
| 380–780 | 4/6 | 67 | 0/6 | 0 | 5/6 | 83 | |
| > 1,000 | 16/18 | 89 | 0/18 | 0 | 18/18 | 100 | |
| Overall | 22/30 | 73 | 0/30 | 0 | 28/30 | 93 | |
Total number of merozoite surface protein 2 (msp 2) alleles, multiplicity of infection, and reproducibility of results from 90 samples processed in duplicate*
| Reproducibility‡ | ||||||||
|---|---|---|---|---|---|---|---|---|
| Filter paper | Extraction method | PCR positive 15 tested | Total msp 2 alleles | Multiplicity of infection† | A | B | C | D |
| * PCR = polymerase chain reaction. | ||||||||
| † Mean ± SE number msp 2 alleles per sample. | ||||||||
| ‡ A = exact match of number and size of alleles for PCR positive samples processed, i.e., extracted in duplicate; B = difference of one allele; C = difference of more than one allele; D = negative results in duplicate. | ||||||||
| § Not estimated due to negative results. | ||||||||
| Whatman 3MM | Methanol | 12 | 21 | 1.40 ± 0.27 | 6 | 5 | 1 | 3 |
| Chelex | 14 | 30 | 2.00 ± 0.29 | 10 | 4 | 0 | 1 | |
| TE buffer | 15 | 30 | 2.00 ± 0.26 | 13 | 2 | 0 | 0 | |
| Schleicher & Schuell | Methanol | 12 | 19 | 1.27 ± 0.21 | 4 | 7 | 1 | 3 |
| Chelex | 0 | § | § | 0 | 0 | 0 | 15 | |
| TE buffer | 15 | 31 | 2.07 ± 0.25 | 7 | 5 | 3 | 0 | |
Authors’ addresses: Sándor Bereczky, J. Pedro Gil, and Anna Färnert, Infectious Diseases Unit, Karolinska Institutet, Karolinska University Hospital, S-171 76 Stockholm, Sweden, Telephone: 46-8-517-75-281, Fax: 46-8-517-76-740, E-mail: sandor.bereczky@medks.ki.se. Andreas Mårtensson, Infectious Diseases Unit, Karolinska Institutet, Karolinska University Hospital, S-171 76 Stockholm, Sweden and Emergency Medicine Unit, Department of Medicine, Kullbergska Hospital, Katrineholm, Sweden.
Acknowledgements: We thank Hanna-Stina Hanson for constructive discussions, Professor Marita Troye-Blomberg and Dr. Johan Strömberg-Nörklit for providing filter paper samples, and Professor Anders Björkman for advice and comments.
Financial support: This work was part of a project supported by the Swedish International Development Cooperation Agency (SAREC).
REFERENCES
- 1↑
Guthrie R, Susi A, 1963. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32 :338–343.
- 2↑
Lindstrom B, Ericsson O, Alvan G, Rombo L, Ekman L, Rais M, Sjoqvist F, 1985. Determination of chloroquine and its desethyl metabolite in whole blood: an application for samples collected in capillary tubes and dried on filter paper. Ther Drug Monit 7 :207–210.
- 3↑
Prior TW, Highsmith WE, Friedman KJ Jr, Perry TR, Scheuerbrandt G, Silverman LM, 1990. A model for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic fibrosis. Clin Chem 36 :1756–1759.
- 4↑
Farnert A, Arez AP, Correia AT, Bjorkman A, Snounou G, do Rosario V, 1999. Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction. Trans R Soc Trop Med Hyg 93 :50–53.
- 5↑
Long GW, Fries L, Watt GH, Hoffman SL, 1995. Polymerase chain reaction amplification from Plasmodium falciparum on dried blood spots. Am J Trop Med Hyg 52 :344–346.
- 6↑
Gil JP, Nogueira F, Stromberg-Norklit J, Lindberg J, Carrolo M, Casimiro C, Lopes D, Arez AP, Cravo PV, Rosario VE, 2003. Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb). Mol Cell Probes 17 :85–89.
- 7
Kain KC, Lanar DE, 1991. Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol 29 :1171–1174.
- 8↑
Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.
- 9↑
Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, Viriyakosol S, 1999. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg 93 :369–374.
- 10↑
Farnert A, Arez AP, Babiker HA, Beck HP, Benito A, Bjorkman A, Bruce MC, Conway DJ, Day KP, Henning L, Mercereau-Puijalon O, Ranford-Cartwright LC, Rubio JM, Snounou G, Walliker D, Zwetyenga J, do Rosario VE, 2001. Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study. Trans R Soc Trop Med Hyg 95 :225–232.
- 11
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN, 1993. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61 :315–320.
- 12↑
Henning L, Felger I, Beck HP, 1999. Rapid DNA extraction for molecular epidemiological studies of malaria. Acta Trop 72 :149–155.