Evaluation of Five Methods Used to Extract Deoxyribonucleic Acid (DNA) From Human Malaria Parasitized Blood Spotted on the Filter Paper
Keywords:malaria, blood spot, extraction, purity, PCR
Efficient deoxyribonucleic acid (DNA) extraction is critical for a good polymerase chain reaction (PCR) performance in molecular diagnostic, genomic and epidemiological studies of malaria. Blood collected on filter paper is a pratical method for this puposes in high parasitemic individuals. In this paper we evaluated five different methods (Commercial kit, Chelex-saponin, microwave, methanol and Tris-EDTA) for DNA extraction. Blood specimens from six asymptomatic Plasmodium sp.-infected patients in Jayapura General Hospital were spotted onto filter paper, dried, and transported for processing. Evaluation of the results of DNA extraction was carried out by measuring yields using spectrophotometry and PCR amplification for mitochondrial gene. Results showed that the best results of PCR obtanied only with commercial kit method, whereas very low quality of PCR by microwave and no amplified DNA for three other methods. The purity of DNA for all methods was low. The sensitivity of detection for kit was approximately 100% for P. falciparum, P. malariae and P. vivax, whereas microwave method, even though resulted in high concentration of DNA, did not or slightly show the effectiveness in DNA amplification that may due to high inhibitor contents. Even though chelex method was widely used by many malaria laboratories, in this experiment we did not found its effectiveness. We concluded that commercial extraction kit method is well-suited for the DNA extraction in PCR-based assays of malaria.
CDC, Malaria Parasites, 2010.
World Health Organization. World Malaria Report 2005. WHO, Geneva, 2005.
Malkin E, Dubovsky F, Moree M, â€œProgress towards the development of malaria vaccinesâ€, Trends in Parasitology, Vol. 22, No.7, pp.292â€“295, 2006.
World Health Organization, Malaria Fact Sheet, Geneva, 2011.
Simanjuntak CH and Arbani PR, â€œSituasi Malaria di Indonesiaâ€, Cermin Dunia Kedokteran, Vol. 55, pp. 3-4, 1994.
Bojang KA, Milligan PJ, Pinder M, Vigneron L, Allouche A, Kester KE, Ballou WR, Conway DJ and Reece WH, â€œEfficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trialâ€, Lancet, Vol. 358, pp. 1927-1934, 2001.
Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP, Doolan DL, Sacci J, de la Vega P, Dowler M, Paul C. et al., â€œProtection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoitesâ€, J. Infect. Dis. Vol. 185, pp.1155 -1164, 2002.
Luke TC and Hoffman SL, â€œRationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccineâ€, The Journal of Experimental Biology, Vol. 206, pp. 3803-3808, 2003.
Okell LC, Bousema T, Griffin JT, Ouedraogo AL, Ghani AC, Drakeley CJ, â€œFactors determining the occurrence of submicroscopic malaria infections and their relevance for controlâ€, Nat Commun Vol. 3, pp. 1237, 2012.
Bereczky S, Martensson, A, Gil P, FÃ¤rnert A, â€œShort report: rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparumâ€, Am J Trop Med Hyg., Vol. 72, pp. 249-51, 2005.
Cox- Singh J, Mahayet S, Abdullah MS, Singh B, â€œIncreased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extractionâ€, Int J Parasitol., Vol. 27, pp. 1575-1577, 1997.
Yang S and Rothman RE, â€œPCR-based diagnostics for infectious diseases:uses, limitations, and future applications inacute-care settingsâ€, The Lancet Infectious Disease, Vol. 4, pp. 337-348, 2004.
Fredericks DN and Relman DA, â€œApplication of polymerase chain rection to the diagnosis of infectious diseasesâ€, Clin Infect Dis, Vol. 29, pp. 475-486, 2000.
Louie M, Louie L, Simor AE, â€œThe role of DNA amplification technology in the diagnosis of infectious diseasesâ€, CMAJ, Vol. 163, pp. 301-309, 2000.
Farooq U, Dubey ML, Shrivastava SK, and Mahajan RC, â€œGenetic polymorphism in Plasmodium falciparum: Differentiation of parasite isolates of high & low virulence by RAPDâ€, Indian J Med Res., Vol. 136, No. 2, pp. 292â€“295, 2012.
Glasel JA, â€œValidity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratiosâ€, Biotechniques, Vol. 18, pp. 62â€“63, 1995.
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, Thaithong S, Brown KN, â€œHigh sensitivity of detection of human malaria parasites by the use of nested polymerase chain reactionâ€, Mol Biochem Parasitol, Vol. 61, pp. 315-320, 1993.
Farnert A, Arez AP, Correia AT, Bjorkman A, Snounou G, do Rosario V, â€œSampling and storage of blood and the detection of malaria parasites by polymerase chain reactionâ€, Trans R Soc Trop Med Hyg, Vol. 93, pp. 50â€“53, 1999.
Reza A, â€œForensic implications of PCR inhibitionâ€”A reviewâ€, Forensic Science International: Genetics, Vol. 6, No. 3, pp. 297â€“305, 2012.
Saini HS, Shepherd M and Henry RJ, â€œMicrowave extraction of total genomic DNA from barley grains for use in PCRâ€, Journal of the Institute of Brewing, Vol.105, No. 3, pp. 185â€“190, 1999.
Kain KC and Lanar DE, â€œDetermination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole bloodâ€, Journal of Clinical Microbiology, Vol. 29, No. 6, pp. 1171-1174, 1991.
Canier L, Khim N, Kim S, Sluydts V, Heng S, Dourng D, Eam R, Chy S, Khean C, Loch K, Ken M, Lim H, Siv S, Tho S, Masse-Navette P, Gryseels C, Uk S, Van Roey K, Grietens KP, Sokny M, Thavrin B, Chuor CM, Deubel V, Durnez L, Coosemans M, MÃ©nard D, â€œAn innovative tool for moving malaria PCR detection of parasite reservoir into the fieldâ€, Malaria Journal, Vol. 12, p. 405, 2013.
Morris U, Aydin-Schmidt B, Shakely D, MÃ¥rtensson A, JÃ¶rnhagen L, Ali AS, Msellem MI, Petzold M, Gil JP, Ferreira PE and BjÃ¶rkman A, â€œRapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicabilityâ€, Malaria Journal, Vol. 12, p. 106, 2013.
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