American Journal of Zoology

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DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients

Received: 20 May 2018    Accepted: 01 August 2018    Published: 28 August 2018
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Abstract

The study aim to evaluate guanidine hydrochloride (HCL) as DNA extraction method from Giardia Cyst in human fecal samples and PCR detection using triose phosphate isomerase gene (tpi). Atotal of 30positive fecal samples of Giardia were collected from Hospitals and health centers from three states of Sudan (Khartoum, Gazeira and Sennar) during the periods from June to September 2017 by Convenient sampling method the samp!es were purifid in Phosphate buffer saline , treated with Liquid Nitrogen and boiling, then guanidine hydrochloride method was used for DNA extraction 1ml of sample added to 1ml lysis buffer ,1ml guanidine hydrochloride , 300µl ammonium acetate, and 10µl proteinase K and incubated in 37 C over night, DNA was harvested, confirmed and evaluated by Nanodrops can, and spectrophotometr for concentration, then followed by PCR detection, using ready prepared commercial product (Maxime PCRPriMex (i-Taq 20µl) Macro gene Company, Korea). In the reaction 1.5µl forwards and 1.5µl reverse primer were add to 5µl DNA template and complete the volume to 20µl with sterile D. W. the reaction was conducted in 94°C for 5min as initial temperature 94°C for 30 sec, 55°C for 45 sec, 72°C for 2min, 72°C for The result found that DNA concentration was 34.5000±2.1213ng/m 62.4000±0.56569ng/ml 4.6800±1.46803ng/ml for Khartoum, Sennar, and Gazeira states while PCR only 5samples were+ve 2 (20%) samples Khartoum and 3 (30%) samples Sennar states but no+ve PCR result in Gazeira state. which confirmed by agarose gel electrophoresis,the sdudy conclude that chemical methods of DNA extraction for examples guanidine HCL is not less effective than other, and PCR it is high sensitive to inhibitors. Also it is possible to obtain DNA from G.cyst after treated with heat and liquid nitrogen.

DOI 10.11648/j.ajz.20180101.15
Published in American Journal of Zoology (Volume 1, Issue 1, September 2018)
Page(s) 24-27
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This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

DNA Extraction, G. Lamblia, G. Cyst, Guanidine HCL, PCR Detection, Sennar

References
[1] Adam, R. D. A. Biology of Giardia lamblia. Clin. Microbiol. 2001. Rev. 14:447–475.
[2] Bridget S. Fisher, Carlos E. E srano, Judith A, Cole Modeling Long–Term Host cell–Giardia lamblia interaction in an invitro Co-culture system. PLOS ONE (wwwplosone.org) 2013, V8, ISSUE 12.
[3] Lane, S., and D. Lloyd.. Current trends in research in to the water borne parasite Giardia. Crit. Rev. Microbiol. 2002, 28. 123–147.
[4] WHO. 1996. The world health report 1996. Fighting disease, fostering development. World Health Organization, Geneva, Switzerland.
[5] Smith, H. V., S. M. Caccio`, A. Tait, J. Mc Lauchlin, and R. C. Thompson. 2006. Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends Parasitol. 22:160–167.
[6] Randi M., Mc Cuin R. M. & ClancyL. Modifications to United States Environmental Protection Agency methods 1622and1623for detection of Cryptosporidium oocysts and Giardia cysts in water. Applied and Environmental Microbiology, 2003, 69, 267-274.
[7] Sun C. H., Mc Caffery J. M., Reiner D. S. & Gillin F. D. Mining the Giardia lamblia genome for new cyst wall proteins. Journal of Biological Chemistry, 2003, 278, 21701-21708.
[8] Monis PT, And rews RH, Mayr hofer G, Ey PL. Molecular systematic of the parasitic protozoan Giardia intestinalis. MolBiolEvol1999; 16:1135–44.
[9] Smith, H. V., S. M. Caccio`, A. Tait, J. McLauchlin, and R. C. Thompson. 2006. Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends Parasitol. 22:160–167.
[10] ThompsonRCA. The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Veterinary Parasitol 2004; 126:15–35.
[11] Nash TE, Mc Cutchan T, Keister D, Dame JB, Conard JD, Gillin FD. Restriction endo nuclease analysis of DNA from 15Giardia isolates obtained from humans and animals. J Infect Dis1985; 152:64–73.
[12] Homan WL, van Enckevort FHJ, Limper L, van Eys GJJM, Schoone GJ, Kasprzak W, et al. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol Res1992; 78:316–23.
[13] Maryhofer G, And rews RH, Ey PL, Chilton NB. Division of Giardia isolates from humans in to two genetically distinct assemblages by electrophoretic analysis of enzymes coded at 27loci and comparison with Giardia muris. Parasitology1995; 111:11–7.
[14] Guy R. A., Payment P., Krull U. J. & Horgen P. A._ Real-Time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Applied and Environmental Microbiology, 2003, 69 (9), 5178-5185.
[15] Skotarczak B. Methods for parasitic protozoans detection in the environmental samples. Parasite, 2009, 16, 1-8.
[16] Lujan H. D., Mowatt M. R. & Nash T. E. Mechanisms of Giardia lamblia differentiation in to cysts. Microbiology and Molecular Biology Review, 1997, 61, 294-304.
[17] Lujan H. D., Mowatt M. R., Conrad J. T., Bowers B.& Nash T. E. Identification of anovel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly in to the cyst wall. Journal of Biological Chemistry, 1995, 270, 29307-29313.
[18] AdamskaM., Leońska DuniecA., MaciejewskaA., SawczukM. & SkotarczakB. (2010) Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and Taq Man realtime PCR, Parasite, v, 17, p299-305, http://www.parasite-journal.org.
[19] Harris J. R. & Petry F. Cryptosporidium parvum: structural components of the oocyst wall. Journal of Parasitology, 1999, 85 (5), 839-849.
[20] Jiang J., Alderisio K. A., Singh A. & Xiao L. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Applied and Environmental Microbiology, 2005, 71 (3), 1135-1141.
[21] Carranza P. G., Feltes G., Ropolo A., Quintana S. M., Touz M. C. & Lujan H. D. Simultaneous expression of different variant specific surface proteins in single Giardia lamblia trophozoites during encystation. Infection and Immunity, 2002, 70 (9), 5265-5268.
[22] Sprong, H., Caccio, S. M., vander Giessen, J. W. B., on behalf of the ZOOPNET net work and partners, 2009.Identification of zoonotic assemblages of Giardia duodenalis. PLo SNegl. Trop. Dis. 3, e558, http://dx.doi.org/10.1371/journal.pntd.0000558.
[23] Irshad M. Sulaiman, Ronald Fayer, Caryn Bern, Robert H. Gilman, James M. Trout, Peter M. Schantz, Pradeep Das, Altaf A. Lal, and Lihua Xiao, (2003) Triose phosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis, Emerging Infectious Diseases, www.cdc.gov/eid., Vol. 9, No. 11.
[24] Choy Seow Huey, Mohammed A. K. Mahdy, Hesham M. Al-Mekhlafi, Nabil A. Nasr, Yvonne A. L. Lim, Rohela Mahmud, Johari Surin (2013) Multi locus genotyping of Giardia duodenalis in Malaysia, Infection, Genetics and Evolution journal, Elsevier, www.elsevier.com.
[25] Andrews RH, Adams M, Boreham PF, Mayrhofer G, MeloniBP. Giardia intestinalis: electrophoretic evidence for aspecies complex. IntJ Parasitol 1989; 19:183–90.
[26] Caccio S. M., Ryan, U.,. Molecular epidemiology of giardiasis. Mol. Biochem. Parasitol. 2008, 160, 75–80.
[27] Caccio`, S. M., R. C. Thompson, J. McLauchlin, and H. V. Smith. 2005. Unravelling Cryptosporidium and Giardia epidemiology. TrendsParasitol. 21:430–437.
[28] Covacin, C., Aucoin, D. P., Elliot, A., Thompson, R. C.,. Genotypic characterization of Giardia from domestic dogs in the USA. Vet. Parasitol. 2011, 177, 28–32.
[29] Harris J. R. & Petry F. Cryptosporidium parvum; structural components of the oocyst wall. Journal of Parasitology, 1999, 85 (5), 839-849.
[30] HomanWL, van Enckevort FHJ, LimperL, van Eys GJJM, Schoone GJ, Kasprzak W, et al. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNAprobes. ParasitolRes1992; 78:316–23.
[31] Anas M. Elnazeer, Khitma Hassan Elmalik, Ahmed A. Elshikh- Isolation, excystation and in vitro Culture of Giardia-spp from fecal samples of suspected patients in RPMI media EUROPEAN ACADEMIC RESEARCH Vol. III, Issue 10/ January 2016.
Author Information
  • International University of Africa (IUA), Department of Microbiology, Faculty of Pure and Applied Sciences, Molecular Biology Research Lab Supervisor (IUA), Khartoum, Sudan

  • National University Research Institute (NURI), Khartoum, Sudan

  • National University Research Institute (NURI), Khartoum, Sudan

  • Tropical Medicine Research Institute, National Research Centre, Khartoum, Sudan

  • Faculty of Veterinary, Khartoum University Medicine, Khartoum, Sudan

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  • APA Style

    Anas Mahadi Elnazeer, Abdallha Elssir, Rihab Ali Omer, Mubark Mustafa, Khitma Hassan Elmalik. (2018). DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients. American Journal of Zoology, 1(1), 24-27. https://doi.org/10.11648/j.ajz.20180101.15

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    Anas Mahadi Elnazeer; Abdallha Elssir; Rihab Ali Omer; Mubark Mustafa; Khitma Hassan Elmalik. DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients. Am. J. Zool. 2018, 1(1), 24-27. doi: 10.11648/j.ajz.20180101.15

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    AMA Style

    Anas Mahadi Elnazeer, Abdallha Elssir, Rihab Ali Omer, Mubark Mustafa, Khitma Hassan Elmalik. DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients. Am J Zool. 2018;1(1):24-27. doi: 10.11648/j.ajz.20180101.15

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  • @article{10.11648/j.ajz.20180101.15,
      author = {Anas Mahadi Elnazeer and Abdallha Elssir and Rihab Ali Omer and Mubark Mustafa and Khitma Hassan Elmalik},
      title = {DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients},
      journal = {American Journal of Zoology},
      volume = {1},
      number = {1},
      pages = {24-27},
      doi = {10.11648/j.ajz.20180101.15},
      url = {https://doi.org/10.11648/j.ajz.20180101.15},
      eprint = {https://download.sciencepg.com/pdf/10.11648.j.ajz.20180101.15},
      abstract = {The study aim to evaluate guanidine hydrochloride (HCL) as DNA extraction method from Giardia Cyst in human fecal samples and PCR detection using triose phosphate isomerase gene (tpi). Atotal of 30positive fecal samples of Giardia were collected from Hospitals and health centers from three states of Sudan (Khartoum, Gazeira and Sennar) during the periods from June to September 2017 by Convenient sampling method the samp!es were purifid in Phosphate buffer saline , treated with Liquid Nitrogen and boiling, then guanidine hydrochloride method was used for DNA extraction 1ml of sample added to 1ml lysis buffer ,1ml guanidine hydrochloride , 300µl ammonium acetate, and 10µl proteinase K and incubated in 37 C over night, DNA was harvested, confirmed and evaluated by Nanodrops can, and spectrophotometr for concentration, then followed by PCR detection, using ready prepared commercial product (Maxime PCRPriMex (i-Taq 20µl) Macro gene Company, Korea). In the reaction 1.5µl forwards and 1.5µl reverse primer were add to 5µl DNA template and complete the volume to 20µl with sterile D. W. the reaction was conducted in 94°C for 5min as initial temperature 94°C for 30 sec, 55°C for 45 sec, 72°C for 2min, 72°C for The result found that DNA concentration was 34.5000±2.1213ng/m 62.4000±0.56569ng/ml 4.6800±1.46803ng/ml for Khartoum, Sennar, and Gazeira states while PCR only 5samples were+ve 2 (20%) samples Khartoum and 3 (30%) samples Sennar states but no+ve PCR result in Gazeira state.  which confirmed by agarose gel electrophoresis,the sdudy conclude that  chemical methods of DNA extraction for examples guanidine HCL is not less effective than other, and PCR it is high sensitive to inhibitors. Also it is possible to obtain DNA from G.cyst after treated with heat and liquid nitrogen.},
     year = {2018}
    }
    

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  • TY  - JOUR
    T1  - DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients
    AU  - Anas Mahadi Elnazeer
    AU  - Abdallha Elssir
    AU  - Rihab Ali Omer
    AU  - Mubark Mustafa
    AU  - Khitma Hassan Elmalik
    Y1  - 2018/08/28
    PY  - 2018
    N1  - https://doi.org/10.11648/j.ajz.20180101.15
    DO  - 10.11648/j.ajz.20180101.15
    T2  - American Journal of Zoology
    JF  - American Journal of Zoology
    JO  - American Journal of Zoology
    SP  - 24
    EP  - 27
    PB  - Science Publishing Group
    SN  - 2994-7413
    UR  - https://doi.org/10.11648/j.ajz.20180101.15
    AB  - The study aim to evaluate guanidine hydrochloride (HCL) as DNA extraction method from Giardia Cyst in human fecal samples and PCR detection using triose phosphate isomerase gene (tpi). Atotal of 30positive fecal samples of Giardia were collected from Hospitals and health centers from three states of Sudan (Khartoum, Gazeira and Sennar) during the periods from June to September 2017 by Convenient sampling method the samp!es were purifid in Phosphate buffer saline , treated with Liquid Nitrogen and boiling, then guanidine hydrochloride method was used for DNA extraction 1ml of sample added to 1ml lysis buffer ,1ml guanidine hydrochloride , 300µl ammonium acetate, and 10µl proteinase K and incubated in 37 C over night, DNA was harvested, confirmed and evaluated by Nanodrops can, and spectrophotometr for concentration, then followed by PCR detection, using ready prepared commercial product (Maxime PCRPriMex (i-Taq 20µl) Macro gene Company, Korea). In the reaction 1.5µl forwards and 1.5µl reverse primer were add to 5µl DNA template and complete the volume to 20µl with sterile D. W. the reaction was conducted in 94°C for 5min as initial temperature 94°C for 30 sec, 55°C for 45 sec, 72°C for 2min, 72°C for The result found that DNA concentration was 34.5000±2.1213ng/m 62.4000±0.56569ng/ml 4.6800±1.46803ng/ml for Khartoum, Sennar, and Gazeira states while PCR only 5samples were+ve 2 (20%) samples Khartoum and 3 (30%) samples Sennar states but no+ve PCR result in Gazeira state.  which confirmed by agarose gel electrophoresis,the sdudy conclude that  chemical methods of DNA extraction for examples guanidine HCL is not less effective than other, and PCR it is high sensitive to inhibitors. Also it is possible to obtain DNA from G.cyst after treated with heat and liquid nitrogen.
    VL  - 1
    IS  - 1
    ER  - 

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