Back
 AJMB  Vol.10 No.4 , October 2020
Evaluation of DNA Extraction Methods for Detection of Leishmania by Polymerase Chain Reaction
Abstract: There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation KitTM). Human peripheral blood samples were inoculated with 104 promastigotes of Leishmania infantum. DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.
Cite this paper: Batalini, L. , Castro, S. , Souto, C. , Neitzke-Abreu, H. and Lima-Junior, M. (2020) Evaluation of DNA Extraction Methods for Detection of Leishmania by Polymerase Chain Reaction. American Journal of Molecular Biology, 10, 265-272. doi: 10.4236/ajmb.2020.104018.
References

[1]   Anversa, L., Tiburcio, M.G.S., Richini-Pereira, V.B. and Ramirez, L.E. (2018) Human Leishmaniasis in Brazil: A General Review. Revista Associacao Medica Brasileira, 64,281-289.
https://doi.org/10.1590/1806-9282.64.03.281

[2]   Neitzke-Abreu, H.C., Venazzi, M.S., Bernal, M.V., Reinhold-Castro, K.R., Vagetti, F., Mota, C.A., Silva, N.R., Aristides, S.M., Silveira, T.G. and Lonardoni, M.V. (2013) Detection of DNA from Leishmania (Viannia): Accuracy of Polymerase Chain Reaction for the Diagnosis of Cutaneous Leishmaniasis. PLoS ONE, 8, e62473.
https://doi.org/10.1371/journal.pone.0062473

[3]   Rodgers, M.R., Popper, S.J. and Wirth, D.F. (1990) Amplification of Kinetoplast DNA as a Tool in the Detection and Diagnosis of Leishmania. Experimental Parasitology, 71, 267-275.
https://doi.org/10.1371/journal.pone.0062473

[4]   Palma, J., Valmorbida, I., da Costa, I.F. and Guedes, J.V. (2016) Comparative Analysis of Protocols for DNA Extraction from Soybean Caterpillars. Genetics and Molecular Research: GMR, 15, 1-9.
https://doi.org/10.4238/gmr.15027904

[5]   Silva, A.O. (2017) Evaluation of Extraction and Purification Protocols of target DNA of Polymerase Chain Reaction in the Leishmania (Viannia) spp. Detection. Dissertation, Aggeu Magalhaes Institut, Oswaldo Cruz Fundation, Recife, Brazil.

[6]   Zorzetto, R., Saraira, L.C., Costa, L., Palma, T.V., Couto, J.C.M., Augusti, L.M., Leal, G.T., Quadros, F.P., Fernandes, G.M., Kolling, J., Nassbaumer, A., Santini, C.S., Schub, G., Sagrilo, M. and Vizotto, B.S. (2015) The PCR Simplex Technique Associated to the Low Cost DNA Extraction Method for Molecular Identification of Shigella spp Clinical Isolates. Disciplinarum Scientia, 16, 11-7.

[7]   Araújo, F.R., Ramos, C.A.N., Luíz, H.L., Péres, I.A.H.F.S., Oliveira, R.H.M., Souza, I.I.F. and Russi, L.S. (2009) Evaluation of a Protocol of Genomic DNA Extraction a From Blood. Technical Notice, 120, 1-5.

[8]   Venazzi, E.A., Roberto, A.C., Barbosa-Tessmann, I.P., Zanzarini, P.D., Lonardoni, M.V. and Silveira, T.G. (2007) Detection of Leishmania (Viannia) DNA in Blood from Patients with American Cutaneous Leishmaniasis. Experimental Parasitology, 115, 399-402.
https://doi.org/10.1016/j.exppara.2006.10.002

[9]   Galluzzi, L., Ceccarelli, M., Diotallevi, A., Menotta, M. and Magnani, M. (2018) Real-Time PCR Applications for Diagnosis of Leishmaniasis. Parasites & Vectors, 11, Article No.273.
https://doi.org/10.1186/s13071-018-2859-8

[10]   Zampieri, R.A. (2013) Comparative Analysis of Molecular Methods for Detection and Identification of Leishmania spp. and Development of Methodology for Leishmaniasis Diagnosis. Dissertation, Institute of Biomedical Science, Universidade de Sao Paulo, Sao Paulo, Brazil.

[11]   Ghaheri, M., Kahrizi, D., Yari, K., Babaie, A., Suthar, R.S. and Kazemi, E. (2016) A Comparative Evaluation of four DNA Extraction Protocols from Whole Blood Sample. Cellular and Molecular Biology (Noisyle-Grand, France), 62, 120-124.

[12]   Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

[13]   Lima-Junior, M.S.C., Zorzenon, D.C.R., Dorval, M.E.C., Pontes, E.R.J.C., Oshiro, E.T., Cunha, R., Andreotti, R. and Matos, M.F.C. (2013) Sensitivity of PCR and Real-Time PCR for the Diagnosis of Human Visceral Leishmaniasis Using Peripheral Blood. Asian Pacific Journal of Tropical Disease, 3, 10-15.
https://doi.org/10.1016/S2222-1808(13)60003-1

 
 
Top