An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis
1
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μL for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.
Abstract
Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μL for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.
참고문헌 (20)
- World Malaria Report, 2015, "World Health Organization", World Health Organization, ~ (2015)
- Coleman RE, "Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum", vivax endemic area in Thailand, 2006, pp.5, 121~
- Ochola LB, "The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard", Lancet Infect Dis, 6, pp.582~588 (2006)[DOI]
- Okell LC, "Factors determining the occurrence of submicroscopic malaria infections and their relevance for control", Nat Commun, 3, pp.1237~ (2012)[DOI]
- Rubio JM, "Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea", Am J Trop Med Hyg, 60, pp.183~187 (1999)[DOI]
- Fontecha GA, "Comparison of molecular tests for the diagnosis of malaria in Honduras", Malar J, 11, pp.119~ (2012)[DOI]
- Lundblom K, "High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA", Malar J, 10, pp.229~ (2011)[DOI]
- Ebrahimzadeh A, "Nested polymerase chain reaction (PCR) on fixed stained slides in comparison to whole blood as a source of DNA in southeast of Iran", J Trop Dis, 2, pp.136~ (2014)
- Port JR, "A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification", Malar J, 13, pp.454~ (2014)[DOI]
- Marshall LA, "Extraction of DNA from malaria-infected erythrocytes using isotachophoresis", Anal Chem, 83, pp.9715~9718 (2011)[DOI]
- Musapa M Kumwenda T, "A simple Chelex protocol for DNA extraction from Anopheles spp", J Vis Exp, 71, pp.1~7 (2013)
- Wang L, "Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated Nal and SDS", Nucleic Acids Res, 22, pp.1774~1775 (1994)[DOI]
- Kho WG, "A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax", Parasitol Int, 52, pp.229~236 (2003)[DOI]
- Rubio JM, "Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the seminested multiplex malaria PCR (SnM-PCR)", Trans R Soc Trop Med Hyg, 1, pp.199~204 (2002)
- Cnops L, "Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR", Malar J, 10, pp.67~ (2011)[DOI]
- Hopkins H, "Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda", J Infect Dis, 208, pp.645~652 (2013)[DOI]
- Robinson T, "Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients", PLoS One, 6, pp.e23204~ (2011)[DOI]
- Morris U, "Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability", Malar J, 12, pp.106~ (2013)[DOI]
- Kritsiriwuthinan K, "Molecular screening of Plasmodium infections among migrant workers in Thailand", J Vector Borne Dis, 48, pp.214~218 (2011)
- Mann R, "Comparative assessment of genomic DNA extraction processes for Plasmodium: identifying the appropriate method", J Vector Borne Dis, 52, pp.273~280 (2015)