Biochemistry & Molecular Biology الكيمياء الحيوية والأحياء الجزيئية
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- ItemGenetic Analysis and Population Structure of Phlebotomus sergenti Sandflies; Vectors of Leishmania tropica in Palestine(Al-Quds University, 2024-05-19) Bushra Abdel Qader Tahboub Al-Amawi; بشرى عبدالقادر طهبوب الأمويPhlebotomus (Ph.) sergenti is a widespread sandfly, it is considered a vector of leishmania tropica in Palestine and other countries; it is the cause of cutaneous leishmaniasis. This study aimed to investigate and analyze the phylogenetic and phylogeographic structure of Ph. sergenti among separated populations in the West Bank; Palestine. Population analysis of these sandflies is necessary for developing models that may provide better understanding of their current geographic distribution. The genetic population structure analysis of fifty Ph. sergenti sandflies were collected from West Bank, Palestine. This study inspected the genetic differentiation between Ph. sergenti populations using a 442 bp fragment of Cyt. b mtDNA gene. Maximum parsimony tree and median joining network were constructed depending on the identified haplotypes. Additionally, one hundred Ph. sergenti sequences previously deposited in the GenBank from ten countries (Iran, Pakistan, Afghanistan, Turkey, Cyprus, Greece, Syria, Lebanon, Morocco and Spain), were included in the study. Nine haplotypes were obtained from the Palestinian populations; six of them were private haplotypes, suggesting the presence of genetic isolation. The global analysis exhibited forty-five global haplotypes and grouped according to their geographical location. Pairwise FST values ranged between 0.58533 and 0.89288, indicating a high genetic differentiation and low genetic flow. Population genetic analysis of Ph. sergenti is considered crucial and may be needed to design an appropriate insecticide spraying programs and for sandfly control models, to restrict the transmission of leishmania parasites in the endemic areas of cutaneous leishmaniasis.
- ItemDevelopment of loop mediated isothermal DNA amplification (LAMP) test for the detection of active brucellosis infections among diary animals(Al-Quds University, 2021-05-29) Ahmad Msallam Ibrahim Makhamreh; أحمد مسلم إبراهيم مخامرهBrucellosis is zoonotic a disease caused by bacterial genus Brucella. It affects a wide range of mammalian species and is transmissible to humans, making it a major socioeconomic problem. Because of the unspecific symptoms and signs that are shared with other febrile diseases, diagnosing brucellosis can be challenging and due to the slow growth rate in blood culture. Rose Bengal test is an internationally recommended test for the screening of brucellosis. They are not always specific as the antibodies can cross-react with other gram-negative bacteria, these tests are not of high value in detecting infection at early stages, May give a false negative result, for a short period after infection, Low sensitivity particularly in long chronic cases, relatively low specificity in endemic areas, since generation and disappearance of antibodies requires some time. To diagnose brucellosis, we developed a LAMP assay. The reaction takes 60 minutes to complete at 63 ° C. Its highly sensitive method , simple, quick, specific, and cost-effective. The strand displacement reaction is carried out at a constant temperature and is characterized by the use of four different primers, each of which is designed to recognize six distinct regions on the target gene. There was no cross-reactivity with the other bacteria. Its More effective than traditional PCR (Less expensive, easier, faster, no hardware required, and with the same accuracy as PCR). The blood test was carried out for 5000 sheep. out of which 200 (4%) were positive. We used the positive results (200) samples and re-examination using the LAMP method that we have developed. Whereas, by developing the LAMP method, we were able to eliminate the interference with other bacteria, and get rid of the false positive result due to the presence of antibodies to Brucella in the blood as a result of a previous infection. Whereas, The LAMP method, unlike the serology approach, is based on the detection of DNA for bacteria rather than antibodies to the bacterium, In addition to removing all of the barriers to detecting brucellosis that existed using serological approaches.
- ItemFollow-up of Tobamovirus infection in cultivated tomato crop using NGS analysis(Al-Quds University, 2024-05-18) Maram Hatem Adel Jaafreh; مرام حاتم عادل جعافرهThe Tomato vegetable plant (Solanum lycopersicum), which bears edible fruit, is a common component of the diet, and it is economically importance across the world. Tobamovirus group (family: Virgaviridae) are destructive agricultural diseases that infect plants. The main goal of the current research is to identify the most tomato cultivars that show the best resistance to viral infections. This outcome could help farmers to avoid many financial losses by choosing specific disease tomato resistance varieties. This study is designed to detect the presence of Tobamovirus relied on next generation sequencing (NGS) technology. The study was performed after collection of leaves, fruit tomato plants and soil samples from 4 different greenhouses located in Jericho district over a period of four months starting from February to May 2023 till the end of the samples collection period, a total of 155 of soil and plant samples were collected. For each collected sample total nucleic acid extraction was done, and processed for genetic analysis. First single stranded cDNA was synthesized from the produced total nucleic acid, followed by amplification via polymerase chain reaction. For each sample two PCR systems were applied for the viral detection and later its DNA sequence analysis was performed by NGS method. All files were uploaded on Galaxy platform program (usegalaxy.org), quality filtered, and analysis were achieved. Among the 155 PCR-tested samples using PCR system 1 and system 2; there was consistency in the PCR results of the two systems. Samples that were seen to have faint or weak amplification were different among the two used PCR systems. It was clear more positive results were seen among the tested samples using PCR system 1 compared to PCR systems 2. From the total tested samples using PCR system 1, 86 samples revealed to have moderate strong PCR results reflected by showing strong PCR bands on agarose gel electrophoresis of Tobamovirus compared to about 46 samples of the same criteria were obtained by PCR system 2. On the other hand, the samples that showed a faint or weak amplification were calculated to be 45 samples applying PCR system 1 and 86 samples applying PCR system 2. All other samples were revealed to be negative by the two used PCR systems. IV Using NGS analysis, the sequences showed 99% similarity to different isolates of Tomato brown rugose fruit virus including the Jordanian tomato Tobamovirus isolate (TBRFV-Jo), (GenBank accession no. KT383474.1).
- ItemFirst molecular detection and genotyping of Neospora caninum in naturally infected cattle and sheep in Palestine(Al-Quds University, 2024-05-05) Heba suleiman salem alfarajeen; هبة سليمان سالم الفراجينNeosporosis has become one of the most common diseases causing abortion in dairy cattle globally. The clinical signs of Neospora caninum have been reported in sheep, goats, deer, and horses. Fetal abortion induced by N. caninum is a common reproductive problem that causes significant economic loss in cattle and sheep husbandry. This study aimed to detect and identify N. caninum in intermediate hosts such as cattle and sheep from Palestine using polymerase chain reaction (PCR) followed by DNA Sanger sequencing and to study the genetic diversity of the study samples, targeting the MS10 microsatellite, using phylogenetic analysis. A total of 124 brain tissue samples were collected from 106 (85%) cattle and 18 (15%) sheep from a slaughterhouse in Jericho-Palestine. The PCR technique was used to identify the Neospora DNA in the brain samples based on the Nc-5 gene. N. caninum was detected in the brain samples; out of 124 samples, 30 (24.19%) samples were positive for N. caninum. The frequency of N. caninum in cattle (25.47%) was higher than that of sheep (16.66%). The infection caused by N. caninum was confirmed by DNA Sanger sequencing of ( 13 ) random positive samples. BLAST analyses of the Nc-5 gene revealed more than 92% to 99% matching with N. caninum sequences available in GenBank. Microsatellite MS10 repeats were used to study the genetic diversity of the Palestinian isolates. The microsatellite genotyping was done by next-generation sequencing (NGS). It was applied to 30 positive samples, and successful results were achieved in 15 samples. Our study sequences displayed 100% similarity with N. caninum isolated from dogs in Liverpool and 99.34% similarity with N. caninum isolated from cattle in Japan. The phylogenetic analysis of 15 N. caninum samples from Palestine and 83 microsatellite (MS10) repeats obtained from different countries and different hosts showed that N. caninum isolates are genetically diverse and distributed into v two main clusters. Locally, the Palestinian isolates are distributed in two clusters and share the same repeats with samples from Asia and Europe. The present study provided the first estimate of the frequency of N. caninum in cattle and sheep from Palestine. Additionally, it was also the first time for the investigation of the phylogenetic analysis of N. caninum based on microsatellite markers.
- ItemDetection of Familial Hypercholesterolemia Variants in Selected Patients with Premature Coronary Artery Disease from Hebron Region(Al-Quds University, 2024-05-15) Enas Yousef Mustafa Sarahna; ايناس يوسف مصطفى سراحنهBackground: The Familial hypercholesterolemia disorder is prevalent but varies across ethnicities. Familial hypercholesterolemia is an autosomal dominant disease with 87% penetrance caused by pathogenic variants in the genes involved in cholesterol metabolism: LDL Receptor, apolipoprotein B or Proprotein Convertase Subtilisin/ Kexin 9 genes, resulting in impaired clearance of circulating Low Density Lipoprotein cholesterol, given that up to 90% of genetically confirmed familial hypercholesterolemia have LDL Receptor variants. It is a highly atherogenic metabolic disorder characterized by lifelong vascular exposure to LDL-C, leading to premature coronary artery disease. Purpose: This work aims to identify the pathogenic variant that cause this disease and verify cascade screening among the studied family. Methods: We selected a patient who fulfilled our inclusion criteria: (1) fasting plasma LDL-C level >190 mg/dL and triglycerides < 220 mg/dL; (2) presence of tendon xanthomata/xanthelasma/corneal arcus or premature coronary artery disease or a first degree relative, or a family history of hypercholesterolemia. The proband’s whole blood sample was sent to Whole Exome Sequencing. Then, blood samples were drawn for PCR and Sanger sequencing from his first-degree relatives. Any newfound case was treated as a new index, and his/her first-degree relatives were screened for that variant. Results: Findings showed that our proband has a heterozygous likely pathogenic missense NM_000527.2: c.1210A>G p. (Thr404Ala) variant in exon 9 of LDL Receptor gene. This variant has not yet been submitted to the Clinvar database. Screening results of his first-degree relatives showed that this variant was transmitted from his father (homozygous familial hypercholesterolemia: GG) to all of his brothers (heterozygous familial hypercholesterolemia: AG). First-degree relatives of affected individuals were screened; a pedigree was drawn. Sequence result and LDL-C level was significantly correlated. Conclusion: Familial hypercholesterolemia is underdiagnosed and undertreated in our population, and this increased the number of premature Atherosclerotic cardiovascular disease cases. Cascade screening is a beneficial and cost-effective process for the diagnosis and treatment of familial hypercholesterolemia early in life using lipid-lowering agents in order to decrease the burden of Atherosclerotic cardiovascular disease and prevent premature cardiovascular death among our population. The variant NM_000527.2: c.1210A>G p. (Thr404Ala) is highly suspected to be FH-causing as A>G substitution has a significant correlation with LDL-C mean level.