Vol.5 No.1 – 12 : Gelatin-coated magnetic nanoparticles-based DNA isolation method: A comparison with commercial DNA isolation kits from whole blood

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By: Kelly S Levano1,2, Luis Jaramillo-Valverde1,2, Elizabeth Delgado1,2, Heinner Guio1,2*

1INBIOMEDIC Research and Technological Center, Lima, Peru;

 2ALBIOTEC, Lima, Peru;

Abstract

DNA isolation is the first step for most molecular tests, thus indicating the importance of using a reliable and suitable method for this procedure. The use of magnetic nanoparticles for blood DNA isolation offers several advantages including: subjecting little mechanical stress to your sample, obtaining higher quality and quantity of DNA and non-laborious procedures. In this study, we isolate genomic DNA using gelatin-coated magnetic nanoparticles from blood samples and test its use compare with three commercial blood DNA isolation kits.  DNA purity and yield were assessed by measuring absorbance at A260/A280 and by agarose gel electrophoresis. The suitability of the isolated DNA for downstream applications was analyzed by end-point PCR and Sanger sequencing. In this study, gelatin-coated magnetite particles for genomic DNA isolation provide an efficient, simple, and inexpensive method that does not require the use of commercial blood DNA Isolation Kit.

Gelatin-coated magnetic nanoparticles-based DNA isolation method A comparison with commercial DNA isolation kits from whole blood

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Vol.5 No.1 – 11 : Assessment of cell-free DNA for early detection of ovarian cancer in women

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By: Waleed M Fathy,1 Sabah Farouk,2 Gamalat Elgedawy,3 Ayman Abd-Elhakim Abd-Elghany, 4 Amira Ahmed2

1Clinical Pathology Department, Faculty of Medicine, Menufia University, Egypt

2Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University Egypt

3Biochemistry Department, National Liver Institute, Menufia University, Egypt

4Obstetrics and Gynecology Department, General Organization of Teaching Hospital, Egypt

Abstract

Objectives: To assess circulating cell-free DNA’s diagnostic potential in Egyptian women with ovarian cancer. Background: Ovarian cancer (OC), one of the most common cancers worldwide, is the most lethal form of gynecological cancer, but the early detection of ovarian cancer would significantly decrease its mortality rate. Circulating plasma cell-free DNA (cfDNA) is nucleic acids in peripheral blood that originate from cell death caused by injury, apoptosis, and necrosis. Circulating cfDNA is normally found in small amounts in the blood of healthy individuals, although increased cfDNA levels have been reported in patients with various clinical conditions, including infection, inflammation, malignancy, connective tissue diseases, ischemic stroke, myocardial infarction, pregnancy-associated disorders, and hemodialysis. Subjects and Methods: This study was conducted on 50 patients with OC, 25 patients with benign ovary disease (BOD), and 25 apparently healthy women used as a control group. All participants were tested for AFP, HCG, CA125, LDH, and circulating cfDNA, which were measured using real-time quantitative Polymerase chain reaction (RT-qPCR). Results: Circulation cfDNA rises more dramatically between OC cases than in both BOD cases and healthy controls, and the results indicate that circulating cfDNA was significantly higher in the OC group (p < 0.001) than both the BOD and control groups. The receiver operator of characteristics (ROC) curve analysis of circulating cfDNA revealed that, at a cut-off value of > 4.13 (fold expression), the sensitivity and specificity for differentiation of OC cases from non-cancer subjects were 97.3% and 92%, respectively. A significant positive correlation was found between circulating cfDNA and CA 125.Conclusion: Circulating CfDNA might be a biomarker for the early diagnosis of ovarian cancer.


Assessment of cell-free DNA for early detection of ovarian cancer in women. (1)


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Vol.5 No.1 – 10 : Clonal Diversity and High Prevalence of Oxa-23 among Carbapenem Resistant Acinetobacter baumannii Isolates in Egypt

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By : Nevine Fam1, Doaa Gamal1, *Dalia Salem1, Heba Dahroug1, Reham M. Wasfy1, Myriam M. Morcos2.

1Department of Microbiology, Theodor Bilharz Research Institute (TBRI), Giza, Egypt.

2Naval Medical Research Laboratories (NAMRU-3). Cairo, Egypt.

Abstract

Background and Aim: Carbapenem-resistant Acinetobacter baumannii (CRAB) is becoming a global threat especially to hospitalized patients. We aimed to address the magnitude of CRAB causing healthcare-associated infections in patients admitted to a tertiary healthcare hospital in Egypt and to study their genetic and epidemiologic diversity. Materials and Methods: Twenty-six CRAB isolates representing 48% of all Acinetobacter baumannii (A. baumannii) isolated in the study period were identified by microbiological culture methods and verified by presence of bla-oxa51. Antimicrobial susceptibility was tested by disc diffusion and MIC was determined by VITEK2 compact system. Phenotypic expression of metallo-β-lactamases (MBLs) was determined by MBL IP/IPI E-test. Carbapenemase encoding genes were identified by PCR and clonal relatedness was studied by pulsed field gel electrophoresis (PFGE) using Apa1 and PulseNet protocol. Results: All A. baumannii isolates were multi-drug resistant (MDR). Colistin and minocycline showed the highest sensitivities of 100% and 61.1%respectively. MBLs were phenotypically detected in 20/26 (76.9%) of the isolates while blaOXA-23-like was the main carbapenem resistance gene recorded in 61.5% followed by blaNDM-1-like (26.9%) and blaGES-like (7.7%).  PFGE typing showed high diversity as most of the isolates were < 80% similar. Conclusion: Carbapenem resistance among A. baumannii isolates is increasing dramatically in our geographic region. BlaOXA-23-likeis the most common gene in CRAB isolates in our hospital setting. In addition, blaNDM-1-like and blaGES-like harboring A. baumannii isolates are exhibiting a considerable spread in hospital environment in Egypt. The clonal diversity of our CRAB isolates suggests that it could be due to horizontal dissemination of mobile genetic elements rather than propagation of a certain clone.

Clonal-Diversity-and-High-Prevalence-of-Oxa-23-among-Carbapenem-Resistant-Acinetobacter-baumannii-Isolates-in-Egypt

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Vol.5 No.1 – 9 : Oxidative stress and DNA damage in Nile Tilapia (Oreochromis niloticus) as biomarkers of aquatic pollution

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By: EL-Hassan Mokhamer, Eman H. Radwan and Moataz Elsaka

Zoologlogy Department, Faculty of science, Damanhour university,Egypt

 (Email: elhassan.gaber@sci.dmu.edu.eg)

Abstract

The main purpose of this study is to evaluate the impact of heavy metals (Pb, Zn, Cu, Fe, and Cd) aquatic pollution of EL-Mahmoudeyia canal on the antioxidant enzymatic activities, GSH content and lipid peroxidation levels (MDA) in Oreochromis. niloticus muscles tissues collected from two areas EL-Mahmoudeyia canal as Polluted area and  Rosetta branch of river Nile as reference area in summer 2018 and winter 2019 as well as DNA damage was assessed in fish gills(erythrocytes) samples by applying comet assay. EL-Mahmoudeyia canal exposed to excessive of industrial effluents which impact the living organisms especially fish. The herein results showed that higher concentrations of heavy metals (Pb, Zn, Cu, Fe, and Cd) were detected in water and fish samples collected from the polluted area in comparison with the reference area, especially in winter.  The accumulation patterns of heavy metals in muscles of O. niloticus, were in the following order: Fe > Zn >Pb> Cu and Cd.The antioxidant enzymatic activities of (SOD, CAT, GPx and GST) and the lipid peroxidation biomarker MDA levels in muscles of O. niloticus collected from the polluted area were found to be significantly increased compared to that of the reference area.Meanwhile, there was a significant decrease in the GSH content levelin muscles of O. niloticus collected from the polluted area compared to that of the reference area. A significant elevation in DNA damage frequencies was observed in fish collected from the polluted areas compared with those from the reference area.These noticeable alterations in the selected antioxidant enzymatic activities in muscles of the O. niloticus go in parallel with the remarkable elevation in the levels of the detected heavy metals in water from EL Mahmoudeyia canal, as a result of pollution in these areas. This study explored the utility of the DNA damage, the altered antioxidant enzymatic activities, GSH content and MDA level as biomarkers of aquatic pollution.

 

Oxidative-stress-and-DNA-damage-in-Nile-Tilapia-Oreochromis-niloticus-as-biomark

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Vol.5 No.1 – 8 : Optimization of Microwave technique conditions for Shrimp chitin deacetylation by response surface methodology

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By : Samar Mohamed Mahdy

Department of Food Science, Faculty of Agriculture, Ain Shams University, Cairo, Egypt

E-mail: samar_mahdy@yahoo.com

Abstract

The objective of this study was to optimize chitosan production conditions from shrimp waste using the response surface methodology corresponding to the degree of deacetylation. The influence of alkaline concentration, microwave power, time and chitin concentration on the N-deacetylation of shrimp wastes chitin by microwave irradiation was investigated. Experimental conditions varied from 30 to 50% NaOH, 1125 to 2450 MHz, 6 to 18 min and 4.76 to 8.30% chitin concentration. The degree of deacetylation increased significantly with increasing time, NaOH conc. , microwave power and decreasing chitin conc. response surface analysis indicated the maximal degree of deacetylation to occur at 50% NaOH, 2400 MHz, 4.76% chitin conc. and 13 min to obtain chitosan with DDA 90.2%. 

The utility of biomarkers in the discrimination of acute coronary syndrome patients

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