DNA Extraction and PCR Detection of G.Lamblia Cyst from Human Fecal Samples in Some Sudanese Suspected Patients
American Journal of Zoology
Volume 1, Issue 1, September 2018, Pages: 24-27
Received: May 20, 2018; Accepted: Aug. 1, 2018; Published: Aug. 28, 2018
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Authors
Anas Mahadi Elnazeer, International University of Africa (IUA), Department of Microbiology, Faculty of Pure and Applied Sciences, Molecular Biology Research Lab Supervisor (IUA), Khartoum, Sudan
Abdallha Elssir, National University Research Institute (NURI), Khartoum, Sudan
Rihab Ali Omer, National University Research Institute (NURI), Khartoum, Sudan
Mubark Mustafa, Tropical Medicine Research Institute, National Research Centre, Khartoum, Sudan
Khitma Hassan Elmalik, Faculty of Veterinary, Khartoum University Medicine, Khartoum, Sudan
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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.
Keywords
DNA Extraction, G. Lamblia, G. Cyst, Guanidine HCL, PCR Detection, Sennar
To cite this article
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, American Journal of Zoology. Vol. 1, No. 1, 2018, pp. 24-27. doi: 10.11648/j.ajz.20180101.15
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Copyright © 2018 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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